Design, synthesis and use of synthetic nucleotides comprising charge mass tags

ABSTRACT

Embodiments of the present disclosure relate generally to reporter compositions which are synthetic nucleotides that comprise nucleotides with a high charge mass moiety attached thereto via a linker molecule. The linker molecules can vary in length in part to enable the high charge mass moiety to extend out from a DNA polymerase complex so that polymerization may not be influenced.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation-in-Part application of U.S.application Ser. No. 14/522,185, filed Oct. 23, 2014, which is aContinuation application of U.S. application Ser. No. 13/062,206, filedMar. 3, 2011 that has been issued to U.S. Pat. No. 8,871,921 on Oct. 28,2014, which is the national phase under 35 U.S.C. §371 of prior PCTInternational Application No. PCT/IB2009/007025 which has anInternational filing date of Sep. 3, 2009, designating the United Statesof America, which claims the benefit of U.S. Provisional PatentApplication No. 61/094,025 filed on Sep. 3, 2008, the disclosures ofwhich are hereby expressly incorporated by reference in their entiretyand are hereby expressly made a portion of this application.

BACKGROUND

1. Field

Some embodiments of the present disclosure relate generally to syntheticnucleotides that comprise nucleotides with a charge mass reportermolecule via a long linker molecule. The linker molecules can vary inlength in part to enable the reporter moiety to extend out from the DNAPolymerase complex so that some aspects of polymerization may not beinfluenced entirely or partially.

2. Description of the Related Art

A nucleotide can be defined as a phosphate ester of a nucleoside,comprising a purine or pyrimidine base linked to ribose, or deoxyribosephosphates. The purine nucleotides having chiefly Adenine (A) or Guanine(G) as the base, the pyrimidine nucleotides Cystine (C), Thymoine (T) orUracil (U), and which are the basic repeating units in DNA and RNA(Henderson's dictionary of biological terms, 1989).

DNA is a long polymer comprising units of deoxyribose nucleotides andRNA is a polymer of ribose nucleotides. This sequence of nucleotidebases can determine individual hereditary characteristics.

The central dogma of molecular biology generally describes the normalflow of biological information: DNA can be replicated to DNA, thegenetic information in DNA can be ‘transcribed’ into mRNA, and proteinscan be translated from the information in mRNA, in a process calledtranslation, in which protein subunits (amino acids) are brought closeenough to bond, in order (as dictated by the sequence of the mRNA &therefore the DNA) by the binding of tRNA (each tRNA carries a specificamino acid dependant on it's sequence) to the mRNA.

SUMMARY

A reporter composition is disclosed in accordance with embodiments ofthe present invention. The reporter composition comprises a nucleotideor its derivative, a linker molecule, which may be attached to thenucleotide or its derivative, and a high charge mass moiety, whichcomprises a charge mass that is sufficient to change a property of asensitive detection nanostructure or nano- or mico-sensor operablycoupled to the reporter composition. In some embodiments, the nucleotideor its derivative present in the reporter composition may be selectedfrom the group consisting of a deoxyribonucleotide, a ribonucleotide, apeptide nucleotide, a morpholino, a locked nucleotide, a glycolnucleotide, a threose nucleotide, any synthetic nucleotides, anyisoforms thereof, and any derivatives thereof.

In some other embodiments, the linker molecule comprises a molecule ofthe following general formula, H₂N-L-NH₂, wherein L may comprise alinear or branched chain comprising one or more selected from the groupconsisting of an alkyl group, an oxy alkyl group, an alcohol group, acarboxyl group, an aromatic group, a naphthalene group, an amine group,an amide group, any derivatives thereof, and any combination thereof. Lin the linker may comprise a linear or branched chain comprising one ormore selected from the group consisting of an alkyl group, an oxy alkylgroup, an alcohol group, a carboxyl group, an aromatic group, anaphthalene group, an amine group, an amide group, any derivativesthereof, and any combination thereof and a number of the functionalgroup in the linear chain is 1 to 100, 1 to 75, 1 to 50, 1 to 25 or 1 to1000 in various examples. In some examples, the number of the functionalgroup in the linear or branched chain can be more than 1000. The linkermolecule and/or the high charge mass moiety is configured not to affectnucleotide polymerization by a polymerase and also be removable. Thelinker molecule can be linked to a phosphate group, sugar group or baseof the nucleotide or its derivative.

In some embodiments, the high charge mass moiety present in the reportercomposition can be positive or negative, and further the charge mass ofmoiety can be variable depending on pH. In some examples, the highcharge mass moiety may comprise an aromatic and/or aliphatic skeleton,wherein the skeleton comprises one or more of a tertiary amino group, analcohol hydroxyl group, a phenolic hydroxy group, and any combinationsthereof. In some examples, the high charge mass moiety comprises one ormore of the following groups, any derivatives thereof, and anycombinations thereof:

In addition, the number of the foregoing groups, any derivativesthereof, and any combinations thereof present in the high charge massmoiety can be 1 to 10, 11 to 50, 51 to 100, or more than 100 in variousembodiments.

In one aspect of the invention, a reporter composition may comprise thefollowing molecule:

wherein, R is selected from the group consisting of adeoxyribonucleotide, a ribonucleotide, a peptide nucleotide, amorpholino, a locked nucleotide, a glycol nucleotide, a threosenucleotide, any synthetic nucleotides, any isoforms thereof, and anyderivatives thereof.

In another aspect of the invention, a reporter composition may comprisethe following molecule:

wherein, R is selected from the group consisting of adeoxyribonucleotide, a ribonucleotide, a peptide nucleotide, amorpholino, a locked nucleotide, a glycol nucleotide, a threosenucleotide, any synthetic nucleotides, any isoforms thereof, and anyderivatives thereof.

In still another aspect of the invention, a reporter composition maycomprise the following molecule:

wherein, R is selected from the group consisting of adeoxyribonucleotide, a ribonucleotide, a peptide nucleotide, amorpholino, a locked nucleotide, a glycol nucleotide, a threosenucleotide, any synthetic nucleotides, any isoforms thereof, and anyderivatives thereof.

A kit for determining a nucleotide sequence, comprising the reportercomposition comprising a nucleotide or its derivative, a linkermolecule, and a high charge mass moiety is also disclosed in accordancewith embodiments of the present invention.

A method of synthesizing the reporter composition is also disclosed. Themethod comprises: generating a first covalent linkage between thenucleotide or its derivative and a first amine group of the linker,wherein a phosphate group, a sugar or a base of the nucleotide or itsderivative is linked to the first amine group of the linker; andgenerating a second covalent linkage between a second amine group of thelinker and any functional group present in the high charge mass moiety;wherein the linker comprises at least two amine groups is also disclosedin connection with the present application. In some embodiments, thenucleotide or its derivate used in the method may comprises amonophosphate group. In some other embodiments, the nucleotide or itsderivate used in the method may be selected from the group consisting ofadenosine monophosphate (AMP), guanosine monophosphate (GMP), cytidinemonophosphate (CMP), thymidine monophosphate (TMP), and uridinemonophosphate (UMP).

In still another aspect, a reporter composition comprising a nucleotideor its derivative, a high charge mass moiety comprising an aromatic oraliphatic skeleton, comprising one or more charged groups selected fromthe group consisting of a tertiary amino group, a carboxyl group, ahydroxyl group, a phosphate group, a phenolic hydroxy group, anyderivatives thereof, and any combinations thereof, wherein the one ormore charged groups comprise a charge mass that is sufficient togenerate a detectable change in a property of a sensitive detectionnanostructure or nano-/micro-sensor operably coupled to the reportercomposition, and a linker molecule attached to the nucleotide or itsderivative and the high charge mass moiety, wherein the linker moleculecomprises a linear or branched chain comprising one or more selectedfrom the group consisting of an alkyl group, an oxy alkyl group, analcohol group, a carboxyl group, an amine group, an amide group, anaromatic group, and a naphthalene group, any derivatives thereof, andany combinations thereof is provided.

In some embodiments, the nucleotide in the reporter composition isselected from the group consisting of a deoxyribonucleotide, aribonucleotide, a peptide nucleotide, a morpholino, a locked nucleotide,a glycol nucleotide, a threose nucleotide, and a synthetic nucleotide,and any isoforms thereof.

In some other embodiments, the high charge mass moiety in the reportercomposition comprises one or more selected from the group consisting ofthe following compounds 1-5, any derivatives thereof, and anycombinations thereof:

In certain embodiments, the linker molecule in the reporter compositionis photocleavable or chemically cleavable. In some of certainembodiments, the chemically cleavable linker molecule comprises one ormore selected from the group consisting of: 4-Nitrophenyl2-(azidomethyl)benzoate, 7-Hydroxy-4-methylcoumarinyl2-(azidomethyl)benzoate, any derivatives thereof, and any combinationsthereof.

In alternative embodiments, the photocleavable cleavable linker moleculecomprises one or more selected from the group consisting of thefollowing compounds 6-10, any derivatives thereof, and any combinationsthereof:

In some other embodiments, a number of an alkyl group, an oxy alkylgroup, an alcohol group, a carboxyl group, an amine group, am amidegroup, an aromatic group, and a naphthalene group, any derivativesthereof, and any combinations thereof in the linear or branched chain ofthe linker is 1 to 1000.

In still some other embodiments, the linker molecule or the high chargemass moiety in the reporter composition is configured not to affectnucleotide polymerization by a polymerase.

In still some other embodiments, the high charge mass moiety in thereporter composition is configured to extend out from a nucleotidepolymerase complex.

In still some other embodiments, the linker molecule or the high chargemass moiety in the reporter composition is configured to protrude outfrom a nascent chain so as to reach-down toward the sensitive detectionnanostructure or sensor.

In still some other embodiments, a net charge mass of the high chargemass moiety in the reporter composition is positive or negative, or isvariable depending on pH.

In still some other embodiments, a net charge mass of the high chargemass moiety in the reporter composition is positive in turn in an acidicpH.

In still some other embodiments, a net charge mass of the high chargemass moiety in the reporter composition is negative in turn in analkaline pH.

In still some other embodiments, the linker molecule and/or the highcharge mass moiety in the reporter composition is configured to beremovable.

In still some other embodiments, the reporter composition comprises oneor more selected from the group consisting of the following compounds,any derivatives thereof, and any combinations thereof:

In addition, a kit for determining a nucleotide sequence, comprising thereporter composition in any of the embodiments discussed above is alsodisclosed in accordance with embodiments of the present invention.

Also, a method of synthesizing the reporter composition in any of theembodiments discussed above is also disclosed. The method may comprisegenerating a first covalent linkage between the nucleotide and a firstfunctional group of the linker, wherein a phosphate group, a sugar or abase of the nucleotide is linked to the first functional group of thelinker, and generating a second covalent linkage between a secondfunctional group of the linker and any functional group present in thehigh charge mass moiety.

In some embodiments of the method of synthesizing the reportercomposition, the nucleotide comprises a triphosphate group, selectedfrom the group consisting of adenosine triphosphate (ATP), guanosinetriphosphate (GTP), cytidine triphosphate (CTP), thymidine triphosphate(TTP), and uridine triphosphate (UTP). In some other embodiments, thephosphate group of the nucleotide comprises thiophosphate orphosphoramidate.

BRIEF DESCRIPTION OF DRAWINGS

The patent or application filed contains at least one drawing executedin color. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows a plot current over time of nanowire response to buffer,dATP (deoxyandenosinetriphosphate) and dTTP*(deoxythymidinetriphosphate).

FIG. 2 shows a result from LCMS analysis done with3-(hydroxymethyl)naphthalen-2-ol derivative.

FIG. 3A shows results from a primer extension assay and FIG. 3B showsresults from a melt analysis.

FIG. 4 shows fluorescence observed on the surface after a single Cy3dCTP was incorporated into SCTTP.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The sequencing of the human genome and the subsequent studies have sincedemonstrated the great value in knowing the sequence of a person's DNA.The information obtained by genomic DNA sequence analysis can provideinformation about an individual's relative risk of developing certaindiseases (such as breast cancer and the BRCA 1&2 genes). Furthermore,the analysis of DNA from tumors can provide information about stage andgrading.

Infectious diseases, such as those caused by viruses or bacteria alsocarry their genetic information in nucleotide polymer genomes (eitherDNA or RNA). Many of these have now been sequenced, (or enough of theirgenome sequenced to allow for a diagnostic test to be produced) and theanalysis of infectious disease genomes from clinical samples (a fieldcalled molecular diagnostics) has become one of important methods ofsensitively and specifically diagnosing disease.

Measurements of the presence or absence, as well as the abundance ofmRNA species in samples can provide information about the health statusof individuals, the disease stage, prognosis and pharmacogenetic andpharmacogenomic information. These expression arrays are fast becomingtools in the fight against complex disease and may gain in popularity asprices begin to fall.

In short, the analysis of nucleotide polymers (DNA & RNA) has becomeimportant in the clinical routine, however, cost remains a barrier towidespread global adoption. One reason for this is the complexity of theanalysis requiring expensive devices that are able to sensitivelymeasure up to four different fluorescence channels as RT-PCR experimentsprogress. The cheaper alternatives may require skilled technicians torun and interpret low-tech equipment, such as electrophoresis gels, butthis too may be expensive and a lack of skilled technicians indeveloping countries is prohibitive.

To solve this, a method of nucleotide polymer analysis that may requirecheap and easy to use devices may be required. Some embodiments of thepresent disclosure describes chemical reagents, synthetic nucleotides,that can generally be utilized in such devices. Various embodiments usedin connection with of the present disclosure describes novel syntheticnucleotides that comprises at least some standard nucleotides (or anymodifications, or isoforms), with a high negative charge mass reportermoiety attached via a linker molecule (for instance, attached to the 5′phosphate group), with the linker length of such a length so as toprotrude from a polymerase complex during polymerization, so as not tocause a significant deleterious effect on the polymerase's action.

As used in various embodiments herein, a nucleotide can be, but notlimited to, one of the following compounds, Adenine, Guanine, Cytosine,Thymine, Uracil, and Inosine as well as any modified nucleotides, anynucleotide derivatives and any degenerate base nucleotides. Somenon-limiting examples of such nucleotide may comprise adeoxyribonucleotide, a ribonucleotide, a peptide nucleotide, amorpholino, a locked nucleotide, a glycol nucleotide, a threosenucleotide, any synthetic nucleotides, any isoforms thereof, and anyderivatives thereof. Furthermore, single stranded deoxyribose nucleicacid (ssDNA) can generally be a single stranded nucleotide polymermolecule, comprising Nucleotides and double stranded deoxyribose nucleicacid (dsDNA) can generally be a double strand comprising two ssDNAmolecules linked together via, for example, hydrogen bonding, in areverse complimentary orientation.

Nucleotides can generally be synthesized through a variety of methodsboth in vitro and in vivo. This can involve salvage synthesis (there-use of parts of nucleotides in resynthesizing new nucleotides throughbreakdown and synthesis reactions in order to exchange useful parts), orthe use of protecting groups in a laboratory. In the latter case, apurified nucleoside or nucleobase can be protected to create aphosphoramidite, and can be used to obtain analogues not present innature and/or to create an oligonucleotide.

In some embodiments, nucleotide synthesis comprises the formation of anucleoside (the nitrogenous base joined to a sugar). The sugar involvedin the synthesis and structure of a nucleotide may be either ribose ordeoxyribose; in the latter case, the prefix ‘deoxy’ may be added beforethe name of the nucleoside in all cases except Uracil. A functionalgroup of phosphate can be then esterified to the sugar, creating anucleotide. The phosphate group may comprise one, two, or threephosphates, forming mono-phosphates, di-phosphates, or tri-phosphates,respectively.

Some other embodiments of the present disclosure describe the design,synthesis and use of special synthetic nucleotides comprising anucleotide and a reporter moiety, in which the reporter moiety may notact as a polymerase enzyme blocking moiety attached via a linker.

A reporter moiety or reporter composition used in various embodiments inconnection with the present inventions is a molecule or molecules thatare easily detected by a biosensor or other detection method (such as byeye) and are attached to biomolecules, or probes, or primers that detector amplify molecules of interest.

A linker molecule used in various embodiments is a polymer made up ofmore than one subunit that links a reporter molecule to a nucleotide. Anexample of a linker molecule is a Di-amine linker H₂N-L-NH₂, where Lrepresents a number of further subunits.

As such, the present disclosure should be considered to include allconfigurations that include any nucleotide or its derivative with alinker molecule attaching a reporter moiety with an overall high charge,sufficient enough to get a detectable change in a sensitive biosensorthat can detect small variations in charge mass at or near its surface.Accordingly several examples presented in this application are presentedonly for the purpose of illustration and should not be considered tolimit the scope of the invention.

In various embodiments, the synthetic nucleotides can have at least someof the following aspects:

-   -   1. The reporter moieties reports based upon its charge mass, not        enzymatic activity, fluorescence etc;    -   2. Each synthetic nucleotide may either carry the same charge        mass reporter moiety, or carry a different charge mass;    -   3. The reporter moieties may be easily cleaved; and/or    -   4. The nucleotides may be cheaply and easily mass synthesized.

There are several possible positions available for the attachment oflinkers and the reporter moieties. It is important to attach the linkerso as to not interfere with polymerization or hydrogen bonding betweenthe bases of nucleotides when hybridizing with its compliment base inanother nucleotide polymer (i.e. when two strands of reverse complimentDNA hybridize to form a double stranded DNA molecule), One possibleposition can be the phosphate linkage in the nucleotide. Furthermore, byattaching the linker to the 5′ position in the phosphate, it will blockfurther nucleotide additions as it will prevent phosphodiester bondformation.

Another possible positions available for the attachment of linkers andthe reporter moieties, is on either the sugar group or the base group.

Some other embodiments describe methods of the use in which the linkerand reporter moiety can be cleaved from the synthetic nucleotide in theiterative manner after detection. As one of the possible places toattach the linker is the 5′-phosphate end of phosphate group, which willprevent further nucleotide additions then, by cleaving the linker willtherefore remove this block and allow for further nucleotide additions.

As way of an example, there are at least two options available whichcould facilitate synthesis at the 5′-phosphate terminal:

-   -   1. Thiophosphate; and/or    -   2. Phosphoramidate.

The proposed linker therefore can have the following structure at leastin some embodiments:

H₂N-L-NH₂

where, L could be, but is not limited to, any linear or branched chainmolecule that is configured to link to a nucleotide as well as a highcharge mass moiety, both of which are present in a synthetic nucleotide.In some embodiments, L comprises a plurality of an alkyl group, an oxyalkyl group or the combination thereof with various lengths. In oneembodiment, the number of an alkyl group, an oxy alkyl group or thecombination thereof in L is 1 to 100. In another embodiment, the numberof an alkyl group, an oxy alkyl group or the combination thereof in L is1 to 75. In still another embodiment, the number of an alkyl group, anoxy alkyl group or the combination thereof in L is 1 to 50. In stillembodiment, the number of an alkyl group, an oxy alkyl group or thecombination thereof in L is 1 to 25. In some other embodiments, thenumber of an alkyl group, an oxy alkyl group and the combination thereofin L can be more than 100. While NH₂ is presented for the purpose ofillustration, this NH₂ can be substituted with any other function groupthat can be cross-linked to a nucleotide or its derivative as well as ahigh charge mass moiety, both of which are present in a syntheticnucleotide. Some illustrative examples that can be used instead of NH₂include, but not limited to, any alkyl group (e.g. C_(n)H_(2n+1),wherein n represents a positive integer number such as 1, 2, 3, andetc), any alcohol group (e.g. C_(n)H_(2n)OH, wherein n represents apositive integer number such as 1, 2, 3, and etc), any carboxyl group(e.g. COOH), any amide group (e.g. CONH), and any derivatives thereof.As the linker molecules can vary in length and chemical structure inpart to enable the reporter moiety to extend out from a nucleotidepolymerase (e.g. DNA polymerase, RNA polymerase and others) complex sothat some aspects of polymerization may not be influenced entirely orpartially

The easy access to the linkers of various lengths can be considered as abenefit in a situation where the desired length of the linker may not beknown completely or partially. This may make the optimizationexperiments easy.

The linker with the nucleotide (say Adenosine as an illustrativeexample) therefore may have the following structure at least in someembodiments. While adenosine is presented in some examples below, thisadenosine can be substituted with any other natural or syntheticnucleotide, any modifications thereof and any derivatives thereof insome other embodiments.

In some embodiments, various lengths of linkers at this position mayhave the following structures (exemplified with the Adenosine):

1. Ethylenediamine (2 carbon bond length separation)

2. Pentanediamine (5 carbon bond length separation)

3. Length equivalent to 13 carbon bond length separation

Thus in some embodiments, the linkers thus selected can be:

1. Easily available;

2. Easy to link and cleave (please refer the probable protocols below);and/or

3. Not to interact with the polymerase and the polynucleic acid strandand/or not affect nucleotide polymerization and growth of a nascentnucleotide polymer.

The reporter moiety: As used herein a “reporter moiety” is a molecule ormolecules that are easily detected by a biosensor or other detectionmethod (such as by eye) and are attached to biomolecules, or probes, orprimers that detect or amplify molecules of interest that are normallydifficult to detect without the presence of the reporter moiety‘reporting’ on its presence. The reporter moieties can be any chargedmolecule, group of charged molecules and even many charged moleculesarranged dendritically. The reporting mode is their charge, which isdetected by sensitive charge detection biosensors, such asnanowire/nanotube FETS, nanopores and other piezoelectric biosensors. Insome embodiments, the reporter moieties can be associated with the otherproperties like the chromophoric nature for enabling their detection byUV or visible detector or the fluorescent nature making them to bedetected by the fluorimetric detection. Furthermore, the mass of thereporter moiety can be exploited using biosensors that can detect mass,such a surface Plasmon resonance biosensors and cantalivers.

The charge on the reporter: certain embodiments of the present inventiondescribe the reporter moiety to carry a large charge mass. In oneembodiment, the reporter moiety may introduce a higher charge mass tothe synthetic nucleotide than the charge mass of the nucleotide or itsderivative, which is present in the synthetic nucleotide. However, inanother embodiment, the charge mass introduced by the reporter moietycan be substantially equal to or less than the charge mass of thenucleotide or its derivative, which is present in the syntheticnucleotide. Some non-limiting and illustrative examples of a reportermoiety are provided in this specification. These examples are providedonly for the illustration purpose and therefore should not be consideredto limit the scope of the invention. The chemical structure and/ordimension (e.g. length, size, and mass of a molecule used as a reportermoiety) of a reporter moiety may not be restricted as long as thereporter moiety is configured to provide a charge mass to the syntheticnucleotide and also not to affect polymerization reaction of nucleotidespartially or entirely.

The charge on the moiety can be positive or negative. Taking intoconsideration the nature of linkage, the following provides some aspectsof the selection of charge that can be possibly used in some embodimentsof the present disclosure.

In some embodiments, a reporter moiety having a high charge mass,thereby also identified as a high charge mass moiety, may comprise anaromatic or aliphatic skeleton, comprising one or more charged groupsselected from the group consisting of a tertiary amino group, a carboxylgroup, a hydroxyl group, a phosphate group, a phenolic hydroxy group,any derivatives thereof, and any combinations thereof, wherein the oneor more charged groups comprise a charge mass that is sufficient tochange a property of a sensitive detection nanostructure ornano/micro-sensor operably coupled to the reporter composition.

Positive charge: In some embodiments, the large number of positivecharges can generally be induced on the reporter moiety through theincorporation of tertiary amino groups on the aromatic or aliphaticskeleton. In such embodiments, in turn in the acidic pH (less than 7),these groups may acquire the positive charges making them detectable.

Negative charges: In some other embodiments, the negative charges cangenerally be induced on the reporter moiety through the incorporation ofa carboxyl group, a hydroxyl group, a phosphate group, an alcoholhydroxyl group and/or a phenolic hydroxy group (or functionality) on thearomatic or aliphatic skeleton. Given below are some of the proposedreporter moieties which meet the above mentioned criteria. The fragmentslisted below may be available and able to link to the linker through theamino terminal. The additional advantage could be that the reagents thatare proposed for the phosphoramidate linkage formation may be the sameas this amide linkage formation (Therefore reducing costs of the systemfurther).

Moreover, at least in part due to the stability of this linkage to thealkaline pH (above 7), the process of induction of negative charge wouldbe of no or substantially small interference.

For the purpose of illustration, the following three non-limitingexamples are presented. These examples are provided only for the purposeof illustration and therefore should not be considered to limit thescope of the invention. As such, any modifications on the followingexamples are certainly included in the scope of the invention. Forexample, any substitution of one or more groups (e.g. —OH, ═O, COOH, andothers) linked to the examples can be practiced. Also oligomerization orpolymerization of one of more of the following examples can also bepermitted. Further any other chemical structure or molecule with variousdimensions (e.g. length, size, and mass of the reporter moiety) can beused as a reporter moiety if such chemical structure or molecule isconfigured to provide a charged mass to the synthetic nucleotide andalso not to affect polymerization reaction of nucleotides partially orentirely.

After acquiring the charges, some of these reporters in certainembodiments may exist as follows,

whereas, the reporter-1 and reporter-3 may be available on shelf,reporter-2 may be custom synthesized.

The reporter moieties proposed can generally (be) thus:

1. Easily available or synthesizable;

2. Bear a large charge;

3. Not costly; and/or

4. Easy to link and cleave.

Final compounds (monomers): Based on the above propositions, the finalstructures of the nucleotides along with the linkers and the reporterswould be as follows at least in certain parts of embodiments. Thefollowing examples of some final compounds are also provided for thepurpose of illustration and therefore should not be considered to limitthe scope of the invention. As described above, any variations permittedfor a nucleotide or its derivative, a linker and a high charge massreporter moiety are also permitted to a final compound. Thus, for theadenosine as a nucleotide at the 5′-phosphate terminal in some examples,if the linker is, say, C 13 equivalent (option 3 above), the variouslinkers would make the final structures looks as below:

One proposed final synthetic nucleotide-1 (note the reporter is inmonomer form and this can be increased by aggregating these monomers toincrease charge mass as required):

Another proposed synthetic nucleotide-2 (note the reporter is in monomerform and this can be increased by aggregating these monomers to increasecharge mass as required):

Still another proposed synthetic nucleotide-3 (note the reporter is inmonomer form and this can be increased by aggregating these monomers toincrease charge mass as required):

The following is a non-limiting, illustrative example of synthesisprotocols used in at least some embodiments:

-   -   1. Synthesis of 5′-phosphoramidates of Adenosine: (Linkage of        Nucleotide with the diamine linker). Method of Chu et. al. can        be used for synthesizing 5′-amino derivatives of adenosine        phosphoramidate in which diamantes and adenosine monophosphate        (AMP) can be dissolved in water. EDAC was added later on and was        incubated at room temperature with constant stirring. The        reaction was monitored till completion.    -   2. Synthesis of Final proposed structures: (Linkage of the        diamine linker with reporter moiety). Method of Chu et. al. can        be used for synthesizing 5′-amino derivatives of adenosine        phosphoramidate in which diamines and adenosine monophosphate        (AMP) can be dissolved in water. EDAC was added later on and        then incubated at room temperature with constant stirring. The        reaction was monitored till completion.

One advantage of the similar procedure is that it may work out for boththe steps leading to the formation of final compounds as monomers.

In some illustrative examples of some embodiments, (see below) cleavageof the linkers and reporter moieties may need to be done. The linkageslike phosphoramidates can generally be rather readily cleaved by the useof acids like trifluoroacetic acid at an ambient temperature. By way ofan illustrative example, the proposed synthetic nucleotide-2demonstrated as a probable 3D view below. The aromatic ring at thebottom left of the molecule bears three hydroxy functions which couldpotentially get converted to the negative charge under slight alkalineconditions. Following is the 3D conformation of the adenosine attachedwith the Reporter-1 through linker 3 and the related data.

Approximate distance between the phosphoramidate and terminal chargedatom may be about 20 angstroms, which could generally be sufficient toinduce the charge potential in the surface for detection. This distancecan further be altered with the further modifications in the phase atleast in part by changing the linker lengths. The charge on the terminalreporter moieties can also be changed by the variations in the chemistryof reporter moieties.

In one embodiment, the reporter composition recited in the appendedclaims comprises any nucleotide with a cleavable linker moleculeattached to a high charge mass moiety, wherein the synthetic nucleotide(otherwise referred to as the reporter composition) has a charge that issufficient to cause a detectable change in the property of a sensitivedetection nanostructure or nano/micro-sensor, when the reportercomposition is operably coupled to the nanostructure (as for example, byaddition of the synthetic nucleotide (reporter composition) to a nascentchain during a sequencing by synthesis procedure).

In some embodiments, sequencing reactions can be tracked throughmonitoring changing electrical properties of nanowires throughout primerextension. In such an instance, each nucleotide is then added one afteranother and the conductance of the nanowires monitored. When theconductance changes sufficiently (e.g., the change in conductance isdetectable) then a nucleotide has been incorporated into the nascentchain.

In certain embodiments, sequencing reactions may utilize a reportercomposition, e.g., comprising: a nucleotide or its derivative, a highcharge mass moiety comprising an aromatic or aliphatic skeleton,comprising one or more charged groups selected from the group consistingof a tertiary amino group, a carboxyl group, a hydroxyl group, aphosphate group, a phenolic hydroxy group, any derivatives thereof, andany combinations thereof, wherein the one or more charged groupscomprise a charge mass that is sufficient to change a property of asensitive detection nanostructure or nano-/micro-sensor operably coupledto the reporter composition, and a linker molecule attached to thenucleotide or its derivative and the high charge mass moiety, whereinthe linker molecule comprises a linear or branched chain comprising oneor more selected from the group consisting of an alkyl group, an oxyalkyl group, an alcohol group, a carboxyl group, an amine group, amamide group, an aromatic group, and a naphthalene group, any derivativesthereof, and any combinations thereof.

In certain embodiments, it is configured to detect the addition ofindividual nucleotides by sensing electrical charge associated with areporter moiety using a nanostructure that is capable of detecting achange in electrical charge density (e.g., by detecting a change inresistance). In some of such embodiments, a charged moiety (or areporter moiety) has an electrical charge that is sufficient to changean electrical property of a sensitive nanostructure, when the reportercomposition is operably coupled to the sensitive nanostructure. Forexample, when a new nucleotide that may contain a reporter compositionis added to the growing polymer in a sequencing by synthesis reaction,the charge density at, or near the surface of the sensitivenanostructure increases, and this can be detected by a change inproperty in the sensitive detection nanostructure or nano-/micro-sensor.More particularly, if using a nanowire as the detecting structure, atleast in some embodiments, an increase in charge caused by the additionof the reporter (having a charged moiety) may be detected by a change inresistance in the wire, due to a phenomenon called the field effect. Inother words, at least in some embodiments, the claimed reportercomposition, by virtue of its high electrical charge, is capable whencoupled to a sensitive detection nanostructure or nano-/micro-sensor, ofcausing an electrical change (resistance) in the sensitive detectionnanostructure or nano-/micro-sensor. In the detection of a change inresistance, e.g. a change in electrical charge at or near the surface ofthe sensitive nanostructure or sensor, or an innate electrical chargeadded by the newly added nucleotide at or near the surface of thesensitive nanostructure or sensor, the distance of which is dependant onmode of device operation and gate dielectric/fluid interface and forfield effect transistors (FET) falls within a range of 0-100 nm based onthe charge neutralization of oppositely charged species cancelling outthe effect of Coulombic interactions beyond that range—although, aswould be well understood by one skilled in the art, for other indirectdetection methods the range may differ) by the sensitive detection mayoccur therefore when the new nucleotide is added to a growing chain ofthe nucleotide sequence to be sequenced that is located at or near thesurface of the sensitive nanostructure. In certain alternativeembodiments, the distance may be a function of a biosensor in use. Thus,for instance, if the biosensor is a field effect transistor, it could bedependent on a solution and the field effect transistor sensor, and insome of certain cases, the distance can be within a micron of the sensoror within the sensing ragen of the sensor. The detection of a change inresistance can be measured to determine the identity of the newly addednucleotide. In other words, in some embodiments, such a detection doesnot require any additional step, before or after the addition of the newnucleotide, such as removing the previously added one or more nucleotidefrom the growing chain of the nucleotide sequence (or from the reactionzone), removing a compound blocking the addition of the new nucleotideto the growing chain of the nucleotide sequence, and/or converting achemical and/or physical property of the newly added nucleotide(s).Therefore, in some embodiments, the detection can occur instantly orsubstantially immediately after the new nucleotide is added to thereaction zone (or the growing chain of the nucleotide sequence).However, in some alternative embodiments, any additional step, before orafter the addition of the new nucleotide could occur, e.g. removing thepreviously added one or more nucleotide from the growing chain of thenucleotide sequence (or from the reaction zone), removing a compoundblocking the addition of the new nucleotide to the growing chain of thenucleotide sequence, and/or converting a chemical and/or physicalproperty of the newly added nucleotide(s).

In certain embodiments, one or more derivatives of any of thecompositions or compounds disclosed in this application may be utilized.In some of such embodiments, the derivatives may comprise the compoundshaving the same or substantially the same functionality (or activity)compared to its respective original compounds and having one or moresubstitutions in any of elements (e.g. C, O, N, H and more), any offunctional groups (e.g. —OH, ═O, —N₃, —NO₂, —COOH, —CH₃, and more), andany parts of the skeleton or branches of the compounds. An example ofsuch a derivative can be but is not limited to combining reportercompositions 1 and 4 shown below in a range of combinations:

The supercharged nucleotides may have a reporter moiety added to themvia a linker. The linker length and nature can vary, while reportercompositions have a large negative charge. The large negative charge ofthe reporter moiety serves a number of functions. Some illustrative, andnon-limiting examples of reporter moiety compositions (1-5) are shownbelow:

Reporter moieties can amplify the electronic effect of a superchargednucleotide compared to that of an un-modified nucleotide when in closeproximity to a nanowire, enabling a particular base to be called basedon the signature electronic response and it also allows for a longerread length as the charge can be detected further from the nanowiresurface—nanowires can only detect charges close to their surface (thedistance from the nanowire in which charge can be sensed is called theDebye length). FIG. 1 shows the relationship of current with time ofnanowire response to buffer, dATP (deoxyandenosinetriphosphate) anddTTP* (deoxythymidinetriphosphate). For the results shown in FIG. 1,sensing experiments were performed by detecting the real-timeconductance change of the silicon nanowires before and after dNTP(deoxyribonucleotide triphosphate) addition. The change in conductanceis attributed to the introduction of the negatively charged nucleotideson the surface of the nanowires. The graph in the figure illustrates thedetection of equimolar solutions (10 μM) of dATP and the superchargeddTTP, respectively, in a 0.05×PBS buffer solution. The injection of thenegatively charged canonical dATP occurs at 1000s and its detection isdemonstrated with a 16% change in conductance. At 2000s, thenon-canonical supercharged dTTP is injected and the positive shift inconductance (20%) is attributed to the enhanced negative charge of thesupercharged moiety associating towards the nanowire surface. Theseresults demonstrate that it is possible to detect and differentiatebetween two nucleotides carrying discrete negative charges, by thechange in the electrical conductance profile of the nanowires.

As the reaction proceeds, the nucleotides may be added further andfurther from the nanowire surface, which is where linker length maybecome important. And finally, the linker can serve to enable the highcharge mass moiety to extend out from a DNA polymerase complex in orderto avoid or minimize interference with the on-going polymerization. Someillustrative and non-limiting examples of supercharged nucleotides A, G,T and C (SCATP, SCGTP, SCTTP and SCCTP) are shown below:

Once a nucleotide has been incorporated into the nascent chain, the highcharged reporter moiety can be, but not necessarily, removed (cleaved)and the next synthetic nucleotide complete with a reporter moiety isincorporated. For example, there are two types of cleavable linkerssensitive to reductive conditions: disulfide bridges and azo compounds.They are efficiently and rapidly cleaved by mild reducing agents likedithiothreitol (DTT), b-mercaptoethanol or tris(2-carboxyethyl)phosphine(TCEP). Some illustrative and non-limiting examples of cleavablefunctionalities are shown below:

4-Nitrophenyl 2-(azidomethyl)benzoate

7-Hydroxy-4-methylcoumarinyl 2-(azidomethyl)benzoate

Furthermore, numerous photoremovable protecting groups can be applicableand one of such groups includes compounds containing anortho-nitrobenzyl motif (see, e.g. the compound no. 10 below). Knownphotoremovable groups, such as examples 6-10, have not been previouslyused in the context of nucleotides as previously describes. Thephotoremovable protecting group (ppg) can be efficiently removed inaqueous media by irradiation at a wavelength (e.g. 350 nm) avoidingdamage to the nucleotide base.

wherein, R comprises a linear or branched chain comprising of but notlimited by an alkyl group, an oxy alkyl group, hydrocarbon, a hydrazone,a peptide linker, or a combination thereof.

For illustrative purpose, initial testing of3-(hydroxymethyl)naphthalen-2-ol derivative was carried out. Themolecule was dissolved in a mixture of methanol and water, and was thenirradiated for 90 min with light from a mercury lamp, which emits lightwith a wavelength of approximately 250 nm. LCMS analysis showed 50%consumption of the starting material. After a further 90 min, there wasapproximately 20% starting material remaining.

As shown in FIG. 2, a major peak at 5 min was observed in the LCMS by UVdetection, but it did not ionize in either positive or negative mode. ¹HNMR spectroscopy showed the residual starting material along with othersignals that correspond to a second benzylic group corresponding to therelease of the diol. In the experiments illustrated in FIG. 2, themolecule was dissolved in a mixture of methanol and water, and was thenirradiated for 90 min with light from a mercury lamp, which emits lightwith a wavelength of approximately 250 nm. LCMS analysis showed 50%consumption of the starting material. After a further 90 min, there wasapproximately 20% starting material remaining. As seen in the figure,AM-5-124-1 is after 90 mins and AM-5-124-2 is after the further 90 mins.The peak at 7.2 min is starting material and shows as m/z 404 in the MSas it is its sodium salt. The peak at 5 mins may be due to the diolbeing released.

As specified the linker chain can play an important role, at least insome embodiments, in enabling the nucleotide to remain a polymerasesubstrate, as such any modified nucleotides have to be shown to bebiologically active. In order to exemplify this and as an example ofcertain embodiments, a polymerase reaction mix may be used for real-timeprimer extension analysis with Rotor Template, which requires theincorporation of a single SC-TTP nucleotide followed by a sequence of 44dCTP, dGTP, dATP nucleotides.

Primer extension reactions were monitored using the Rotorgene QReal-time thermocycler, this allows primer extension to be monitoredfluorescently using a dsDNA intercalator (SYBR Safe) with secondary meltanalysis to provide further information on product composition. Thismethod can be used with an oligonucleotide primer/template pair designedto allow complete extension of the primer with the incorporation of asingle SC-TTP nucleotide. Initially, the experiment was be conductedwith a 40° C. incubation and then repeated with a 65° C. incubation(optimum synthesis temperature for most commercial DNA Polymerases).Control reactions with standard dNTPs (positive control) and no dNTPs(negative control) were included, alongside a control(s) formis-incorporation. FIG. 3A captures the extension of the primers in thepresence of standard dNTPs (yellow) and also in the reaction containingSCTTP (blue, yellow), although the maximum fluorescence intensityassociated with SC-TTP (blue) incorporation is slightly reduced,indicating a slight reduction in final product concentration. Reactionslacking dTTP or DNA polymerase did not display the equivalent increasein fluorescence, indicating failure of primer extension (red amdpurple). FIG. 3B shows the melt analysis and confirms the variance inend products between the negative (no polymerase, red and purple) andpositive (standard dNTPs, yellow) control reactions with melt peaks at64.3° C. and 80.5° C. respectively. Interestingly, a very weak secondarymelt peak was identified in the No dTTP (GCA), indicating a lowfrequency of mis-incorporation.

Supercharged nucleotide incorporation can be further validated usingaccurate stage motors (Nanomotion, Israel) and a combination ofmicrofluidic spotting devices functionalized areas on Si/SiO₂ wafer. Insome embodiments, very small volumes of material are transferred to asurface via a sharp tip.

In order to demonstrate primer extension optically, SC-TTP wasincorporated into a sequence prior to a Cy3 labelled dCTP. SC-TTP doesnot contain a fluorescent moiety, by incorporating a Cy3 dCTP after theSC-TTP in the sequence; modified SC-TTP incorporation could bedemonstrated (FIG. 4). Conditions for single Cy3 dCTP incorporation wereelucidated using a standard PAGE-gel set-up and transferred to bulksilicon substrates. Competitor oligos, complementary to the templateswere also designed to aid gel analysis of a single stranded product.Primer extension reactions were conducted using the following reactionmix:

2 μl 10X Polymerase Buffer 0.5 μl 800 nM Primer 0.5 μl 600 nM Template 2μl 2.5 mM dNTP mix 2 μl 1000 μ/ml DNA polymerase 13 μl Ultra-pure water

Following incubated at 40° C. in a hot block reactions were terminatedby the addition of a ‘stop buffer’ containing chelators to inhibitpolymerase activity in addition to formamide and competitor oligo toencourage denaturation of the double stranded DNA duplex forelectrophoresis. Interestingly, in reactions containing SC-TTP, bandswere observed with a larger shift than in the positive control reaction.The additional shift is likely a result of the increased molecularweight of the SC-TTP compared to standard dCTP, potentially retardinggel migration. As such, this shift provides evidence towards successfulSC-TTP incorporation.

Sequencing reactions performed on bulk Si/SiO₂ surfaces using both 3′and 5′ surface tethered 30mer DNA, immobilised onto APDMES/PDC modifiedSi/SiO₂ surface in the typical 4×4 array format. Here, Cy3 labelled dCTPwas selectively incorporated to a hybridised primer using the covalentlybound 30mer probe as a template. The reaction mix was spread over thesilicon surface and incubated overnight in a dark humid chamber atambient temperature. The silicon was then rinsed in 1× Thermopol bufferand dried using a nitrogen line. Fluorescence was visualised using anOlympus BX60 and imaged using Axio vision camera and Axiovision 3.1software.

FIG. 4 shows a fluorescence image taken of a printed arraypost-sequencing and wash steps. The very weak fluorescence indicates thesingle base incorporation of Cy3 labelled dCTP. It should be noted thateach spot would contain a number of DNA strands where the primerextension has taken place and therefore the florescence observed is dueto several Cy3 dCTP's not just one base. The control experiment where noprimer was introduced into the reaction mixture yielded no fluorescenceon the surface.

EXAMPLES

The following description is an illustrative example of some embodimentsof the present disclosure.

Example 1 DNA Sequencing

The sequencing methodology in one example may not use fluorescence andexpensive sensitive cameras, but instead may detect the addition of thesynthetic nucleotides described in some aspects of the presentdisclosure, at least in part by sensing the electrical charge ofreporter moiety, using sensitive nanostructures or sensors that may becapable of detecting a build up of charge mass at, or near, theirsurface. When a new nucleotide is added to the growing polymer in asequencing by synthesis reaction, the charge density at, or near thesurface of the sensitive nanostructure may increase and this can bedetected by a change in property in the sensitive detectionnanostructure or nano-/micro-sensor (for instance, if using a nanowire,or carbon nanotube, as the detecting structure, an increase in chargecaused by the addition of a nucleotide close to its surface may bedetected by a change in resistance in the wire, due to a phenomenoncalled the field effect). However, as the polymer grows, the signal maydiminish as the charges carried by the nucleotides being added may betoo far away from the sensitive nanostructure (e.g. nanowire) or sensorto illicit a change in property of the sensitive detection nanostructureor nano-/micro-sensor and no signal may be observed. Therefore, the‘read length’ (amount of sequence data that is able to be obtained bythis method of nucleotide sequencing) can be limited.

As used herein this particular example, a “sensitive detectionnanostructure” can be any structure (nanoscale or not) which can becapable of detecting any change in charge at, or near it's surface andat any point may have at least one cross-sectional dimension less thanabout 500 nanometers, typically less than about 200 nanometers, moretypically less than about 150 nanometers, still more typically less thanabout 100 nanometers, still more typically less than about 50nanometers, even more typically less than about 20 nanometers, stillmore typically less than about 10 nanometers, and even less than about 5nanometers. In other embodiments, at least one of the cross-sectionaldimensions can generally be less than about 2 nanometers, or about 1nanometer. In one set of embodiments the sensitive detectionnanostructure or sensor can have at least one cross-sectional dimensionranging from about 0.5 nanometers to about 200 nanometers. For certainstructures or sensors the detection of a change in charge is not directbut may be indirect i.e. via detection of from an additional property asa function of the fundamental change in charge.

The properties of a sensitive detection nanostructure or sensor maychange in response to surface, or near surface charge in a way that maybe measurable via piezoelectric measurements, electrochemicalmeasurement, electromagnetic measurement, photodetection, mechanical,measurement, acoustic measurement, gravimetric measurement and the like.An example of a sensitive detection nanostructure or sensor may include,but not limited to, two dimension field effect transistors (FET),cantalevers, nanowires (operated as a FET or impedance or otherwise),nanopores, piezoelectric films, carbon nanotubes, and all appropriatemacro-, micro-, nano-, pico-, zepto-, or smaller structures. The sensormay also be a waveguide.

Certain embodiments of the present disclosure may address thislimitation, at least in part by using synthetic nucleotides that maycomprise normal nucleotides, with a high negative (or positive) chargemass reporter moiety attached via a linker molecule (for instance,attached to the 5′ phosphate group), with the linker length increasingas the reaction progresses. This charge mass can be designed to ‘reachdown’ to the sensitive nanostructure (e.g. nanowire) to cause a changein property of the sensitive detection nanostructure ornano-/micro-sensor (e.g. a field effect or other piezo-electric changein the structure depending on the sensitive detection nanostructure ornano-/micro-sensor used). To enable a good quality control measure andto ensure long read lengths by eliminating the build up of many reportermoieties which would cause an ever increasing field effect, thesereporter moieties can be cleaved at least in certain embodiments, toallow for the addition of the next nucleotide in the sequencing bysynthesis sequence.

Therefore, in some embodiments the cyclical reaction may comprise atleast some or whole of the following entire or partial series of events:

-   -   1. The template ssDNA molecule to be sequenced can be either        ligated to the sensitive detection nanostructure or        nano-/micro-sensor and a primer added, bind to a pre-immobilized        primer sequence on the sensitive detection nanostructure or        nano-/micro-sensor, or uncoiled and elongated in a microfluidics        channel arrayed with sensitive detection nanostructure or        nano-/micro-sensors.    -   2. The sensitive detection nanostructure or nano-/micro-sensors        can be washed with water, or a low salt buffer (such as 1×SSC)    -   3. A measure of the sensitive detection nanostructure or        nano-/micro-sensor can be made.    -   4. A mixture containing one synthetic nucleotide, the polymerase        and other elements required for the polymerization reaction can        be added. In one example, if the nucleotide added is        complimentary to the base on the minus strand immediately after        the primer sequence, it may be incorporated into the growing        chain by the polymerase.    -   5. The reaction can then be washed with either water or a low        salt buffer (such as 1×SSC).    -   6. A measure of the sensitive detection nanostructure or        nano-/micro-sensor can be made which can observe the effect        caused by the high charge mass of the reporter moiety.    -   7. The reporter moiety can then be cleaved (for instance by an        acid solution or enzymatically).    -   8. Points 2 through 7 can be repeated for each of the four        nucleotides. And this can be repeated repeatedly until a clear        signal may degrade.

For some embodiments wherein the template molecule is immobilized to, orbound to a probe that can be in turn immobilized to the sensitivedetection nanostructure or nano-/micro-sensor, the linker lengths thatattach the high charge reporter moiety to the synthetic nucleotides mayincrease to enable the charge to ‘reach down’ to the sensitive detectionnanostructure or nano-/micro-sensor to exert an effect. This may benecessary at least in some embodiments as the growing nucleotide polymermay move the next nucleotide addition site farther and farther from thesensitive detection nanostructure or nano-/micro-sensor as thesequencing by synthesis reaction may progress.

For some other embodiments wherein the template molecules is notimmobilized to the sensitive detection nanostructure ornano-/micro-sensor, or hybridized to a primer/probe that can be in turnimmobilized to the sensitive detection nanostructure ornano-/micro-sensor, and can be instead free or immobilized horizontallyacross a cluster of sensitive detection nanostructure ornano-/micro-sensors, a single linker length can be used for each of thecycle reactions.

Example 2 Primer Extension

In some embodiments, the synthetic nucleotides described in some aspectsof the present disclosure for primer extension experiment wherein thedetection is performed on electrical biosensors (nanowire/nanotube FETs,2D FETS, nanopores, piezo-electric films/surfaces, etc). Primerextension is generally defined as a technique that can map or determinea 5′ end of DNA or RNA. For example, primer extension can be used todetermine the start site of the transcription start site for a gene.This technique generally requires a labelled primer, which iscomplementary to a region near the 3′ end of the target gene. The primeris allowed to anneal to the transcript of the target gene and reversetranscriptase is used to synthesize complementary cDNA to the transcriptuntil it reaches the 5′ end of the transcript. By running the product ona polyacrylamide gel, it can be possible to determine thetranscriptional start site, as the length of the sequence on the gelrepresents the distance from the start site to the labelled primer.During the synthesis of cDNA, the synthetic nucleotides disclosed inthis application can be used and added to the nascent cDNA chain. Theaddition of the specific synthetic nucleic acid (e.g. deoxynucleotidewith Adenine, Guanine, Thymidine, or Cystine) can be detected by ananosensor. The nanosensor, which is further described below can beattached to the primer so that the nascent cDNA chain may be attached tothe nanosensor in some embodiments. Alternatively, in some otherembodiments, the transcript of the target sequence may be attached tothe nanosensor (e.g. nanowires, nanotubes, nanobeads, nanopores,nanogaps and others).

Biosensors

As used in various embodiments, a biosensor is generally a device forthe detection of an analyte that combines a biological component with aphysicochemical detector component. In some embodiments, it may comprisethree parts: 1. the sensitive biological element (biological material(e.g. tissue, microorganisms, organelles, cell receptors, enzymes,antibodies, nucleic acids, etc), a biologically derived material orbiomimic). The sensitive elements can be created by biologicalengineering; 2. the transducer or the detector element (works in aphysicochemical way; optical, piezoelectric, electrochemical, etc.) thattransforms the signal resulting from the interaction of the analyte withthe biological element into another signal (i.e., transducers) that canbe more easily measured and quantified; 3. associated electronics orsignal processors that is primarily responsible for the display of theresults in a user-friendly way. In some other examples, the signalprocessing unit may further comprise one or more of a signal sensingunit, a signal recording unit, a data processing unit, and a datareporting unit.

Nanostructures

As used in various embodiments, a nanowire is an elongated nanoscalesemiconductor which, at any point along its length, has at least onecross-sectional dimension and, in some embodiments, two orthogonalcross-sectional dimensions less than 500 nanometers, preferably lessthan 200 nanometers, more preferably less than 150 nanometers, stillmore preferably less than 100 nanometers, even more preferably less than70, still more preferably less than 50 nanometers, even more preferablyless than 20 nanometers, still more preferably less than 10 nanometers,and even less than 5 nanometers. In other embodiments, thecross-sectional dimension can be less than 2 nanometers or 1 nanometer.In one set of embodiments the nanowire has at least one cross-sectionaldimension ranging from 0.5 nanometers to 200 nanometers. Where nanowiresare described having a core and an outer region, the above dimensionsrelate to those of the core. The cross-section of the elongatedsemiconductor may have any arbitrary shape, including, but not limitedto, circular, square, rectangular, elliptical and tubular. Regular andirregular shapes are included. A non-limiting list of examples ofmaterials from which nanowires of the invention can be made appearsbelow.

Nanotubes are a class of nanowires that may find use in the inventionand, in one embodiment, devices of the invention include wires of scalecommensurate with nanotubes. As used herein, a “nanotube” is a nanowirethat has a hollowed-out core, and includes those nanotubes know to thoseof ordinary skill in the art. A “non-nanotube nanowire” is any nanowirethat is not a nanotube. In one set of embodiments of the invention, anon-nanotube nanowire having an unmodified surface (not including anauxiliary reaction entity not inherent in the nanotube in theenvironment in which it is positioned) is used in any arrangement of theinvention described herein in which a nanowire or nanotube can be used.A “wire” refers to any material having a conductivity at least that of asemiconductor or metal. For example, the term “electrically conductive”or a “conductor” or an “electrical conductor” when used with referenceto a “conducting” wire or a nanowire refers to the ability of that wireto pass charge through itself. Preferred electrically conductivematerials have a resistivity lower than about 10⁻³, more preferablylower than about 10⁻⁴, and most preferably lower than about 10⁻⁶ or 10⁻⁷ohm-meters.

Nanopore generally has one or more small holes in an electricallyinsulating membrane that can be used as a single-molecule detector. Insome cases, it can be a biological protein channel in a high electricalresistance lipid bilayer or a pore in a solid-state membrane. Nanoporeis generally a spherical structure in a nanoscale size with one or morepores therein. According to some aspects, a nanopore is made of carbonor any conducting material.

Nanobead is generally a spherical structure in a nanoscale size. Theshape of nanobead is generally spherical but can also be circular,square, rectangular, elliptical and tubular. Regular and irregularshapes are included. In some examples, the nanobead may have a poreinside.

Nanogap is generally used in a biosensor that consists of separationbetween two contacts in the nanometer range. It senses when a targetmolecule, or a number of target molecules hybridize or binds between thetwo contacts allowing for the electrical signal to be transmittedthrough the molecules.

The foregoing nanostructures, namely, nanowire, nanotube, nanopore,nanobead, and nanogap are described to provide the instant illustrationof some embodiments, and not for limiting the scope of the presentinvention. In addition to the foregoing examples, any nanostructure thathas a nanoscale size and is suitable to be applied to nucleic aciddetection methods and apparatus as disclosed in the application shouldalso be considered to be included in the scope of the invention. Itshould be appreciated that detection can also occur with other nonnano-scale structures such on FET arrays which are in the micron-scaleand discussed further below.

In general, sensing strategies for use with nanostructures ornanosensors to detect molecules and compounds is to sense changes in thecharge at, or near their surfaces, or across a nanogap or nanopore,which cause a measurable change in their properties (such as fieldeffect transistors, nanogaps, or piezoelectric nanosensors) to detect &quantify target nucleic acids (DNA, RNA, cDNA, etc).

Aspects of the invention provide a nanowire or nanowires preferablyforming part of a system constructed and arranged to determine ananalyte in a sample to which the nanowire(s) is exposed. “Determine”, inthis context, means to determine the quantity and/or presence of theanalyte in the sample. Presence of the analyte can be determined bydetermining a change in a characteristic in the nanowire, typically anelectrical characteristic or an optical characteristic. E.g. an analytecauses a detectable change in electrical conductivity of the nanowire oroptical properties. In one embodiment, the nanowire includes,inherently, the ability to determine the analyte. The nanowire may befunctionalized, i.e. comprising surface functional moieties, to whichthe analytes binds and induces a measurable property change to thenanowire. The binding events can be specific or non-specific. Thefunctional moieties may include simple groups, selected from the groupsincluding, but not limited to, —OH, —CHO, —COOH, —SO₃H, —CN, —NH₂, —SH,—COSH, COOR, halide; biomolecular entities including, but not limitedto, amino acids, proteins, sugars, DNA, antibodies, antigens, andenzymes; grafted polymer chains with chain length less than the diameterof the nanowire core, selected from a group of polymers including, butnot limited to, polyamide, polyester, polyimide, polyacrylic; a thincoating covering the surface of the nanowire core, including, but notlimited to, the following groups of materials: metals, semiconductors,and insulators, which may be a metallic element, an oxide, an sulfide, anitride, a selenide, a polymer and a polymer gel. In another embodiment,the invention provides a nanowire and a reaction entity with which theanalyte interacts, positioned in relation to the nanowire such that theanalyte can be determined by determining a change in a characteristic ofthe nanowire.

Microstructures or Microsensor

In some embodiments, a sensor which is similar in the foregoingnanostructures with respect to a shape, structure, and property but hasa different dimension, e.g. micro-scale, can be used. Thus, in someexamples, a microstructure may be in form of an elongated microscalesemiconductor which, at any point along its length, has at least onecross-sectional dimension and, in some embodiments, two orthogonalcross-sectional dimensions less than 500 micrometers, preferably lessthan 200 micrometers, less than 150 micrometers, less than 100micrometers, less than 70, less than 50 micrometers, less than 20micrometers, less than 10 micrometers, less than 5 micrometers, lessthan 4 micrometers, less than 3 micrometers, less than 2 micrometers,and even less than 1 micrometer. In other embodiments, thecross-sectional dimension can be less than 2 micrometers or 1micrometer. In one set of embodiments the microwire has at least onecross-sectional dimension ranging from 0.5 micrometers to 200micrometers. Where microwires are described having a core and an outerregion, the above dimensions relate to those of the core. Thecross-section of the elongated semiconductor may have any arbitraryshape, including, but not limited to, circular, square, rectangular,elliptical and tubular. Regular and irregular shapes are included. Anon-limiting list of examples of materials from which microwires of theinvention can be made appears below. Alternatively or in combination,the microstructure or microsensor can be in any shape or structure, e.g.microtubes, micropores, microbeads, and microgaps that have essentiallysimilar properties of nanostructures in terms of sensing an electricalsignal but have a different dimension, at least in some part(s) of itsstructure, e.g. a cross-section, depth, and/or length, in micro-scale.

Field Effect Transistor (FET)

Field effect generally refers to an experimentally observable effectsymbolized by F (on reaction rates, etc.) of intramolecular coulombicinteraction between the centre of interest and a remote unipole ordipole, by direct action through space rather than through bonds. Themagnitude of the field effect (or ‘direct effect’) may depend on theunipolar charge/dipole moment, orientation of dipole, shortest distancebetween the centre of interest and the remote unipole or dipole, and onthe effective dielectric constant. This is exploited in transistors forcomputers and more recently in DNA field-effect transistors used asnanosensors.

Field-effect transistor (FET) is generally a field-effect transistor,which may use the field-effect due to the partial charges ofbiomolecules to function as a biosensor. The structure of FETs can besimilar to that of metal-oxide-semiconductor field-effect transistor(MOSFETs) with the exception of the gate structure which, in biosensorFETs, may be replaced by a layer of immobilized probe molecules whichact as surface receptors. When target biomolecules hybridize or bind, tothe receptors, the charge distribution near the surface changes, whichin turn modulates current transport through the semiconductor transducer(e.g. nanowire).

Biological Samples

The term sample or biological sample generally refers to any cell,tissue, or fluid from a biological source (a “biological sample”), orany other medium, biological or non-biological, that can be evaluated inaccordance with the invention including, such as serum or water. Asample includes, but is not limited to, a biological sample drawn froman organism (e.g. a human, a non-human mammal, an invertebrate, a plant,a fungus, an algae, a bacteria, a virus, etc.), a sample drawn from fooddesigned for human consumption, a sample including food designed foranimal consumption such as livestock feed, milk, an organ donationsample, a sample of blood destined for a blood supply, a sample from awater supply, or the like. One example of a sample is a sample drawnfrom a human or animal to determine the presence or absence of aspecific nucleic acid sequence.

Nucleic Acid or Oligonucleotide

The terms nucleic acid or oligonucleotide or grammatical equivalentsherein refer to at least two nucleotides covalently linked together. Anucleic acid of the present invention is preferably single-stranded ordouble stranded and will generally contain phosphodiester bonds,although in some cases, as outlined below, nucleic acid analogs areincluded that may have alternate backbones, comprising, for example,phosphoramide (Beaucage et al. (1993) Tetrahedron 49(10):1925) andreferences therein; Letsinger (1970) J. Org. Chem. 35:3800; Sprinzl etal. (1977) Eur. J. Biochem. 81: 579; Letsinger et al. (1986) Nucl. AcidsRes. 14: 3487; Sawai et al. (1984) Chem. Lett. 805, Letsinger et al.(1988) J. Am. Chem. Soc. 110: 4470; and Pauwels et al. (1986) ChemicaScripta 26: 1419), phosphorothioate (Mag et al. (1991) Nucleic AcidsRes. 19:1437; and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu etal. (1989) J. Am. Chem. Soc. 111:2321, O-methylphophoroamidite linkages(see Eckstein, Oligonucleotides and Analogues: A Practical Approach,Oxford University Press), and peptide nucleic acid backbones andlinkages (see Egholm (1992) J. Am. Chem. Soc. 114:1895; Meier et al.(1992) Chem. Int. Ed. Engl. 31: 1008; Nielsen (1993) Nature, 365: 566;Carlsson et al. (1996) Nature 380: 207). Other analog nucleic acidsinclude those with positive backbones (Denpcy et al. (1995) Proc. Natl.Acad. Sci. USA 92: 6097; non-ionic backbones (U.S. Pat. Nos. 5,386,023,5,637,684, 5,602,240, 5,216,141 and 4,469,863; Angew. (1991) Chem. Intl.Ed. English 30: 423; Letsinger et al. (1988) J. Am. Chem. Soc. 110:4470;Letsinger et al. (1994) Nucleoside & Nucleotide 13:1597; Chapters 2 and3, ASC Symposium Series 580, “Carbohydrate Modifications in AntisenseResearch”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al. (1994),Bioorganic & Medicinal Chem. Lett. 4: 395; Jeffs et al. (1994) J.Biomolecular NMR 34:17; Tetrahedron Lett. 37:743 (1996)) and non-ribosebackbones, including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, CarbohydrateModifications in Antisense Research, Ed. Y. S. Sanghui and P. Dan Cook.Nucleic acids containing one or more carbocyclic sugars are alsoincluded within the definition of nucleic acids (see Jenkins et al.(1995), Chem. Soc. Rev. pp. 169-176). Several nucleic acid analogs aredescribed in Rawls, C & E News Jun. 2, 1997 page 35. These modificationsof the ribose-phosphate backbone may be done to facilitate the additionof additional moieties such as labels, or to increase the stability andhalf-life of such molecules in physiological environments.

Sensing Strategies

In one aspect of the invention, a biological material configured to binda nanostructure is a nucleic acids. Such nucleic acids may include DNA,RNA, and any derivatives thereof. In one embodiment, the biologicalmaterial is DNA. When DNA is attached to the nanostructure, the numberof nucleotides may range from 5 bases to 100 bases. In some embodiments,the number of DNA nucleotides may be 7 bases, 10 bases, 15 bases, 20,bases, 25 bases, 30 bases, 35 bases, 40 bases, 45 bases, 50 bases, 60bases, 70 bases, 80 bases, 90 bases and 100 bases. In some otherembodiments, ribonucleic acids and any nucleic acid derivatives may beattached to the nanostructure. In still some other embodiments, DNA, RNAand its derivatives may be used simultaneously. Therefore in oneexample, DNA sequences may be attached to the nanostructure, whereas inanother example, RNA sequences may be attached to the nanostructure,still another example, nucleic acid derivatives such as adeoxyribonucleotide, a ribonucleotide, a peptide nucleotide, amorpholino, a locked nucleotide, a glycol nucleotide, a threosenucleotide, any synthetic nucleotides, any isoforms thereof, and anyderivatives thereof may be attached to the nanostructure. In some otherexamples, nucleotide sequences comprising DNA and RNA, DNA and nucleicacid derivatives, RNA and derivatives, and DNA, RNA and derivatives maybe attached to the nanostructure.

In another aspect of the invention, a nanostructure is conducting andcan sense the electric charge, which is originated from the syntheticnucleotide as disclosed in this application, at its surface, vicinity,inner tubes and/or the pores therein. One of key aspects of anydiagnostic device is the ability to perform accurate detection ofbiomolecules with the performance determined by how well it detectsspecifically (i.e. a low false positive rate) and sensitively (i.e. alow false negative rate). Nanosensors that can sense changes in thecharge at, or near their surfaces, or across a nanogap or nanopore,which cause a measurable change in their properties, at least in partdue to the target molecule binding to a probe immobilized on or near thenanostructures, provide a method for ultra-sensitive detection withoutor with limited use of the need for labels (expensive chemicals that canbe bound to the biomolecule or molecule of interest to enable detectiondevices to ‘sense’ them).

The present disclosure generally relates to molecular biologicalprotocols and sensing strategies for use with nanosensors that maydetect molecules and compounds by sensing changes in the charge at, ornear their surfaces, or across a nanogap or nanopore, which cause ameasurable change in their properties (such as field effect transistors,nanogaps, or piezoelectric nanostructures or nanosensors) to detect &quantify target nucleic acids (DNA, RNA, cDNA, etc). The basic functionof these biosensors may require that a nucleotide polymer probe (orsynthetic nucleotide polymer such as PNA, Morpholinos, etc) beimmobilized on, or near to, the nanostructures and the build up oftarget molecules binding to the probe can cause an increase in chargedensity at or near the surface of the nanostructures or nanosensors, dueto the charge of the probe. For instance, an amplified PCR fragmentbinding to a probe (with a reverse complimentary sequence to the targetnucleotide polymer), immobilized on a nanowire can cause a measurablechange conductance (ΔG) due to the increase in negative charge at, ornear to the nanowire's surface, due to a phenomena called the fieldeffect. In some embodiments, the electric charge present in thesynthetic nucleotide, which is originated from the nucleotide itself andthe high charge mass reporter moiety, can be detected by thenanostructures/nanosensors.

These nanosensors may offer the potential for sensitive and dynamicdetection of biomolecules, however, this sensitivity may bring with it anumber of issues. For instance, natural fluctuations of charge at thesurface, within the sample matrix may cause noise, in part due to theflanking sequences of target nucleotide polymer molecules (i.e. the overhanging sequences that don't bind to the probes). Furthermore, if manytarget molecules are being detected at the same time on an array ofnanosensors, it would be favorable to standardize the size and thereforecharge mass, of each of these molecules to allow for more stringentcomparisons and quality control. Moreover, having a standard size forall target molecules allows for standardization of probe hybridizationconditions in the array assay design.

Biosensor System

Biosensor is generally an analytical device that may convert molecularevents into electrical signals. The nanostructures used in a biosensorare generally used to detect components of interest such as nucleicacids. Biosensors can generally operate in the liquid or gas phase,opening up an enormous variety of applications, e.g., for integrateddevices and for downstream applications. Therefore, the biosensors canbe manufactured inexpensively and portable and are optionally used asimplantable detection and monitoring devices. Alternatively, thebiosensor can be coupled with other high-resolution apparatus such asmass-spectroscopy and provide further information including thedetection of presence, abundance and/or structural variation of thetarget biomolecules.

One aspect of the invention involves a sensing element of a biosensor,which can be an electronic sensing element, and a nanowire able todetect the presence, or absence, of an analyte in a sample (e.g. a fluidsample) containing, or suspected of containing, the analyte. Nanoscalesensors of the invention may be used, for example, in chemicalapplications to detect pH or the presence of metal ions; in biologicalapplications to detect a protein, nucleic acid (e.g. DNA, RNA, etc.), asugar or carbohydrate, and/or metal ions; and in environmentalapplications to detect pH, metal ions, or other analytes of interest.

Another aspect of the invention involves an article of a biosensorcomprising a sample exposure region and a nanowire able to detect thepresence of absence of an analyte. The sample exposure region may be anyregion in close proximity to the nanowire wherein a sample in the sampleexposure region addresses at least a portion of the nanowire. Examplesof sample exposure regions include, but are not limited to, a well, achannel, a microchannel, and a gel. In preferred embodiments, the sampleexposure region holds a sample proximate the nanowire, or may direct asample toward the nanowire for determination of an analyte in thesample. The nanowire may be positioned adjacent to or within the sampleexposure region. Alternatively, the nanowire may be a probe that isinserted into a fluid or fluid flow path. The nanowire probe may alsocomprise a micro-needle and the sample exposure region may beaddressable by a biological sample. In this arrangement, a device thatis constructed and arranged for insertion of a micro-needle probe into abiological sample will include a region surrounding the micro-needlethat defines the sample exposure region, and a sample in the sampleexposure region is addressable by the nanowire, and vice-versa. Fluidflow channels can be created at a size and scale advantageous for use inthe invention (microchannels) using a variety of techniques such asthose described in International Patent Publication No. WO 97/33737,published Sep. 18, 1997, and incorporated herein by reference.

In another aspect of the invention, an article may comprise a pluralityof nanowires able to detect the presence or absence of a plurality ofone or more analytes. The individual nanowires may be differentiallydoped as described above, thereby varying the sensitivity of eachnanowire to the analyte. Alternatively, individual nanowires may beselected based on their ability to interact with specific analytes,thereby allowing the detection of a variety of analytes. The pluralityof nanowires may be randomly oriented or parallel to one another.Alternatively, the plurality of nanowires may be oriented in an array ona substrate.

Where a detector is present, any detector capable of determining aproperty associated with the nanowire can be used. The property can beelectronic, optical, or the like. An electronic property of the nanowirecan be, for example, its conductivity, resistivity, etc. An opticalproperty associated with the nanowire can include its emissionintensity, or emission wavelength where the nanowire is an emissivenanowire where emission occurs at a p-n junction. For example, thedetector can be constructed for measuring a change in an electronic ormagnetic property (e.g. voltage, current, conductivity, resistance,impedance, inductance, charge, etc.) can be used. The detector typicallyincludes a power source and a voltmeter or amp meter. In one embodiment,a conductance less than 1 nS can be detected. In a preferred embodiment,a conductance in the range of thousandths of a nS can be detected. Theconcentration of a species, or analyte, may be detected from less thanmicromolar to molar concentrations and above. By using nanowires withknown detectors, sensitivity can be extended to a single molecule. Inone embodiment, an article of the invention is capable of delivering astimulus to the nanowire and the detector is constructed and arranged todetermine a signal resulting from the stimulus. For example, a nanowireincluding a p-n junction can be delivered a stimulus (electroniccurrent), where the detector is constructed and arranged to determine asignal (electromagnetic radiation) resulting from the stimulus. In suchan arrangement, interaction of an analyte with the nanowire, or with areaction entity positioned proximate the nanowire, can affect the signalin a detectable manner. In another example, where the reaction entity isa quantum dot, the quantum dot may be constructed to receiveelectromagnetic radiation of one wavelength and emit electromagneticradiation of a different wavelength. Where the stimulus iselectromagnetic radiation, it can be affected by interaction with ananalyte, and the detector can detect a change in a signal resultingtherefrom. Examples of stimuli include a constant current/voltage, analternating voltage, and electromagnetic radiation such as light.

Another aspect of the present invention provides an article comprising ananowire and a detector constructed and arranged to determine a changein an electrical property of the nanowire. At least a portion of thenanowire is addressable by a sample containing, or suspected ofcontaining, an analyte. The phrase “addressable by a fluid” is definedas the ability of the fluid to be positioned relative to the nanowire sothat an analyte suspected of being in the fluid is able to interact withthe nanowire. The fluid may be proximate to or in contact with thenanowire.

In some embodiments, the nanostructures can be assembled into aplurality of parallel arrays such as micro-columns at higher densitiesthan is and in a format compatible with currently availablemicro-fluidic systems. The nanostructure arrays optionally comprise aplurality of nanostructures such as nanowires, nanotubes, nanopores,nanobeads, nanogaps, or a combination thereof. Each nanostructure of thearray can be electrically connected, e.g., via two or more electrodes toa battery for applying a voltage across the nanowire and a detector, fordetection of any changes in conductance of the nanowire. Alternatively,each nanostructure separately receives electricity or only a portion ofnanostructures arrayed together may be electrically connected.

A single detector or a combination of detectors is optionally used todetect the signal from the array of nanowires. For example, eachnanowire linked to a probe comprising different target sequence, whichmay be bound to a same or different probe, is optionally detectedseparately, such that a spatial array of a plurality of nanowires can beused to quickly identify, e.g., a plurality of different nucleotidesequences present in a biological sample such as blood. In someexamples, a plurality of patches of nanostructures are prepared in thearray and each patch presents different probes to detect multiple targetsequences in a biological sample. Alternatively, in some other examples,an entire nanostructures present in the array may present same probes,thereby only one target sequence would be tested for its presence,abundance and/or variation in the sequence.

The detection by the nanostructure or nanosensor is generally a changein conductance of the nanostructure or of its environment. The signalcan be expressed in terms of a change in the voltage across thenanostructure, or the current through the nanostructure. Such changesare typically detected electrically, e.g., with a voltmeter and/or acurrent meter. Alternatively, the signal is detected digitally. In oneembodiment, a voltage is applied across a nanostructure, e.g., ananowire, providing a steady state signal. When a binding event occurson the probe attached to the nanostructure, the electric field in thevicinity of the nanostructure changes and the conductance of thenanostructure changes, producing a fluctuation or shift in the steadystate signal. The signal may be detected, electrically or digitally, andprovides real time detection of the event of interest.

Biosensor can also be integrated into a system for detecting a presence,level and/or variation of biomolecules. In one aspect, such system mayinclude an electrical power supply, monitoring system for applying andmeasuring electrical current across the nanostructure element. Inanother aspect, such system may further include data processingcapabilities to enable the programmed operation of the nanostructuresand to receive, store, and provide useful analysis and display of thedata that is obtained. In addition a computer system to process theobtained data as well as additional processor(s) may be integrated intoa biosensor system if desired. The computer system or any additionalelements present in a biosensor system may provide a software(s) foranalyzing the data or for automatic operation and/or manual(s) toperform detection processes with a biosensor. Furthermore, anyadditional elements that may enhance the performance of a biosensorsystem can be added. A biosensor of the present invention can collectreal time data.

Example 3 Hybridization Such as Microarrays

In other embodiments, the synthetic nucleotide disclosed in some aspectsof the present disclosure can be used for hybridization procedures. Somenon-limiting, illustrative examples of the hybridization proceduresinclude a microarray for nucleonic acids as well as proteins. Furtherany other procedures that need hybridization and can determine presence,abundance or any structural variation of the target biomolecules can beincluded.

In one example, the probes used in a microarray can be attached to amedium such as nanosensors (e.g. nanowires, nanotubes, nanobeads,nanoopores, nanogaps, and other nanostructures). In some cases, thetarget nucleotide sequences obtained from the biological samples wouldbe labeled and contacted with the probes. In such cases, the targetnucleotide may incorporate the synthetic nucleotides thereby beinglabeled with “high charge mass”. As such, the binding of the targetsequences to the probes can be readily determined by the nanosensorsthat the probes are attached to. Moreover, the synthetic nucleotidedisclosed in this application can be used, for example, in the methodsof detecting presence, abundance and/or structural variation of nucleicacids as disclosed in the related application of the subjectapplication, U.S. provisional application No. 61/094,017 filed on Sep.3, 2008, the disclosures of which are hereby expressly incorporated byreference in their entirety and are hereby expressly made a portion ofthis application. As such, the use of the synthetic nucleotide of thisapplication is not limited and can be further extended if applicable.

Example 4 Synthesis of Supercharged TTP (SCTTP)5′-O-Dimethoxytrityl-5-iodo-2′-deoxyuridine

5-Iodo-2′-deoxyuridine (5.00 g, 14.1 mmol) was dissolved in anhydrouspyridine (57.5 mL) and stirred under nitrogen. After 5 min,4-(dimethylamino)pyridine (86 mg, 0.70 mmol) and triethylamine (199 mg,275 μL, 1.97 mmol) were added to the reaction mixture, followed by4,4′-dimethoxytriphenylmethyl chloride (5.73 g, 16.9 mmol). Theresulting reaction mixture was stirred at room temperature for 4 h andit was then quenched with methanol (8.3 mL) before being concentratedunder reduced pressure. The residue obtained was partitioned betweenwater (250 mL) and dichloromethane (250 mL). The aqueous phase waswashed with dichloromethane (50 mL) and the organic phases werecombined, dried (MgSO₄) and concentrated under reduced pressure to givea yellow glassy solid. This material was purified using a BiotageIsolera automated chromatography system under normal phase conditions(silica column, gradient of 0→80% ethyl acetate in dichloromethane) withdetection at 254 nm to give 5′-O-dimethoxytrityl-5-iodo-2′-deoxyuridine(8.59 g, 93%), as a pale yellow solid.

R_(f) 0.20 (dichloromethane-ethyl acetate, 8:2, v/v).

¹H NMR (300 MHz, d₆-DMSO): 11.76 (s, 1H, NH), 8.01 (s, 1H, CH), 7.33 (m,9H, 9×ArH), 6.90 (m, 4H, 4×ArH), 6.10 (t, J=7.1 Hz, 1H, CH), 5.32 (d,J=4.5 Hz, 1H, OH), 4.23 (m, 1H, CH), 3.90 (m, 1H, CH), 3.74 (s, 6H,2×OCH₃), 3.17 (m, 2H, CH₂), 2.22 (m, 2H, CH₂).

3′-O-Pivaloyl-5′-O-dimethoxytrityl-5-iodo-2′-deoxyuridine

5′-O-Dimethoxytrityl-5-iodo-2′-deoxyuridine (10.2 g, 15.6 mmol) wasdissolved in anhydrous acetonitrile (205 mL). 4-(Dimethylamino)pyridine(95 mg, 0.78 mmol) and triethylamine (3.95 g, 5.44 mL, 39.0 mmol)followed by trimethylacetic anhydride (5.97 g, 6.50 mL, 32.0 mmol) wereadded. The reaction mixture was stirred and heated at reflux for 20 h.after which it was allowed to cool to room temperature and methanol (6.6mL) was added. The resulting solution was concentrated under reducedpressure to give a brown oil which was purified using a Biotage Isoleraautomated chromatography system under normal phase conditions (silicacolumn, gradient of 0→70% ethyl acetate in dichloromethane) withdetection at 254 nm to give3′-O-pivaloyl-5′-O-dimethoxytrityl-5-iodo-2′-deoxyuridine (9.22 g, 80%),as a white solid. Unreacted 5′-O-dimethoxytrityl-5-iodo-2′-deoxyuridine(2.08 g) was also recovered and was recycled.

R_(f) 0.68 (dichloromethane-ethyl acetate, 8:2, v/v).

¹H NMR (300 MHz, d₆-DMSO): 11.80 (s, 1H, NH), 8.08 (s, 1H, CH), 7.29 (m,9H, 9×ArH), 6.89 (m, 4H, 4×ArH), 6.10 (t, J=7.1 Hz, 1H, CH), 5.21 (m,1H, CH), 3.99 (m, 1H, CH), 3.74 (s, 6H, 2×OCH₃), 3.27 (m, 2H, CH₂), 2.28(m, 2H, CH₂), 1.12 (s, 9H, 3×CH₃).

3′-O-Pivaloyl-5-iodo-2′-deoxyuridine

3′-O-Pivaloyl-5′-O-dimethoxytrityl-5-iodo-2′-deoxyuridine (5.35 g, 7.23mmol) was dissolved in dichloromethane (34 mL) and trifluoroacetic acid(615 μL) was added dropwise. The resulting reaction mixture was stirredat room temperature for 1 h. A second portion of trifluoroacetic acid(615 μL) was added and the mixture was stirred at room temperature for afurther 1 h. A third portion of trifluoroacetic acid (615 μL) was addedand the mixture was stirred at room temperature for 1 h. The resultingmixture was diluted with dichloromethane (120 mL) and washed withsaturated aqueous sodium bicarbonate solution (80 mL). The layers wereseparated and the aqueous phase was extracted with dichloromethane (80mL). The organic layers were combined, dried (MgSO₄) and concentratedunder reduced pressure to afford a pale yellow solid. This solid waspurified using a Biotage Isolera automated chromatography system undernormal phase conditions (silica column, gradient of 0→60% ethyl acetatein dichloromethane) with detection at 285 nm to give3′-O-pivaloyl-5-iodo-2′-deoxyuridine (2.56 g, 81%), as a white solid.

R_(f) 0.41 (dichloromethane-ethyl acetate, 7:3, v/v).

¹H NMR (300 MHz, d₆-DMSO): 11.74 (s, 1H, NH), 8.40 (s, 1H, CH), 6.14 (t,J=7.1 Hz, 1H, CH), 5.33 (t, J=5.0 Hz 1H, OH), 5.20 (m, 1H, CH), 3.97 (m,1H, CH), 3.64 (m, 2H, CH₂), 2.28 (m, 2H, CH₂), 1.16 (s, 9H, 3×CH₃).

3′-O-Pivaloyl-5-(N-Boc-3-amido-propynyl)-2′-deoxyuridine

3′-O-Pivaloyl-5-iodo-2′-deoxyuridine (2.55 g, 5.82 mmol), copper(I)iodide (221 mg, 1.16 mmol) and tetrakis(triphenylphosphine)palladium(0)(670 mg, 0.58 mmol) were dissolved in anhydrous DMF (36.5 mL) and theflask was evacuated and purged with nitrogen. Hunig's base (1.50 g, 1.98mL, 11.6 mmol) and N-Boc-propargylamine (2.71 g, 17.5 mmol) were addedand the resulting mixture was stirred at room temperature overnight. Thereaction mixture was diluted with ethyl acetate (170 mL), washed with 5%aqueous EDTA solution (2×60 mL) and saturated brine (50 mL). The organiclayer was dried (MgSO₄) and concentrated under reduced pressure to givean orange oil. This oil was purified using a Biotage Isolera automatedchromatography system under normal phase conditions (silica column,gradient of 0→100% ethyl acetate in dichloromethane) with detection at295 nm to give 3′-O-pivaloyl-5-(N-Boc-3-aminopropynyl)-2′-deoxyuridine(2.31 g, 85%), as a yellow solid.

R_(f) 0.38 (dichloromethane-ethyl acetate, 1:1, v/v).

¹H NMR (300 MHz, d₆-DMSO): 11.68 (s, 1H, NH), 7.34 (t, J=5.6 Hz, 1H,NH), 8.16 (s, 1H, CH), 6.14 (t, J=7.1 Hz, 1H, CH), 5.27 (t, J=5.0 Hz,1H, OH), 5.19 (m, 1H, CH), 3.95 (m, 3H, CH+CH₂), 3.63 (m, 2H, CH₂), 2.28(m, 2H, CH₂), 1.39 (s, 9H, 3×CH₃), 1.16 (s, 9H, 3×CH₃).

3′-O-Pivaloyl-5-(3-aminopropynyl)-2′-deoxyuridine Trifluoroacetate

3′-O-Pivaloyl-5-(N-Boc-3-aminopropynyl)-2′-deoxyuridine (2.02 g, 4.34mmol) was dissolved in dichloromethane (32 mL) and trifluoroacetic acid(6 mL) was added. The resulting mixture was stirred for 2 h at roomtemperature and was subsequently concentrated under reduced pressure.The residual trifluoroacetic acid was azeotropically removed withdichloromethane (4×50 mL). The material obtained was purified using aBiotage Isolera automated chromatography system under reversed-phaseconditions (C₁₈ column, gradient of 0→60% acetonitrile in 0.1%trifluoroacetic acid) with detection at 287 nm to afford, afterfreeze-drying, 3′-O-pivaloyl-5-(3-aminopropynyl)-2′-deoxyuridinetrifluoroacetate (Fragment 1) (1.55 g, 75%), as an off-white solid.

¹H NMR (300 MHz, d₆-DMSO) 11.80 (s, 1H, NH), 8.28 (br, 4H, CH+NH₃ ⁺),6.14 (t, J=7.1 Hz, 1H, CH), 5.32 (br, 1H, OH), 5.21 (m, 1H, CH), 3.99(m, 3H, CH+CH₂), 3.64 (m, 2H, CH₂), 2.28 (m, 2H, CH₂), 1.16 (s, 9H,3×CH₃).

3-[(1,3)Dioxolan-2-ylmethoxy]benzoic acid ethyl ester

Ethyl 3-hydroxybenzoate (2.23 g, 13.4 mmol), bromoethyl-1,3-dioxolane(8.97 g, 5.56 mL, 53.7 mmol), potassium carbonate (3.72 g, 26.9 mmol)and sodium iodide (0.81 g, 5.37 mmol) were dissolved in anhydrous DMF (6mL) and the reaction mixture was stirred at 120° C. for 17 h. Theresulting suspension was cooled to room temperature and quenched withwater (50 mL). The aqueous phase was extracted with ethyl acetate (3×50mL). The organic layers were combined, washed with water (5×100 mL),dried (MgSO₄) and concentrated under reduced pressure to afford anorange oil. This oil was purified using a Biotage Isolera automatedchromatography system under normal phase conditions (silica column,gradient of 0→35% ethyl acetate in petrol) with detection at 254 nm togive 3-[(1,3)dioxolan-2-ylmethoxy]benzoic acid ethyl ester (3.12 g,92%), as a colorless oil.

R_(f) 0.40 (petrol-ethyl acetate, 75:25, v/v).

¹H NMR (400 MHz, CDCl₃) δ 7.66 (dt, 1H, J=7.5, 1.1 Hz, ArH), 7.59 (dd,1H, J=2.6, 1.5 Hz, ArH), 7.34 (t, 1H, J=7.9 Hz, ArH), 7.14 (ddd, 1H,J=8.3, 2.6, 1.0 Hz, ArH), 5.31 (t, 1H, J=4.1 Hz, CH), 4.36 (q, 2H, 7.2Hz, OCH₂), 4.02 (m, 6H, OCH₂CH₂O+ArOCH₂), 1.39 (t, 3H, J=7.1 Hz, CH₃).

3-[2-Azido-2-(2-hydroxyethoxy)ethoxy]benzoic acid ethyl ester

To a mixture of 3-[(1,3)dioxolan-2-ylmethoxy]benzoic acid ethyl ester(3.12 g, 12.4 mmol) and azidotrimethylsilane (1.57 g, 1.81 mL, 13.6mmol) was added tin(IV) chloride (206 mg, 93 μL, 0.79 mmol). Thereaction mixture was stirred at room temperature for 2 h. The resultingmixture was diluted with 2% aqueous methanol (20 mL) and stirred for 30min before being concentrated under reduced pressure. The residue wasazeotropically dried with ethanol (2×15 mL) to afford a colorlessviscous oil. This oil was purified using a Biotage Isolera automatedchromatography system under normal phase conditions (silica column,gradient of 0→70% ethyl acetate in petrol) with detection at 254 nm togive 3-[2-azido-2-(2-hydroxyethoxy)ethoxy]benzoic acid ethyl ester (2.46g, 69%), as a colorless oil.

R_(f) 0.40 (petrol-ethyl acetate, 53:47, v/v).

¹H NMR (300 MHz, CDCl₃) δ 7.69 (dt, 1H, J=7.7, 1.2 Hz, ArH), 7.59 (dd,1H, J=2.6, 1.5 Hz, ArH), 7.36 (t, 1H, J=8.0 Hz, ArH), 7.13 (ddd, 1H,J=8.3, 2.7, 1.0 Hz, ArH), 4.89 (t, 1H, J=5.1 Hz, CH), 4.38 (q, 2H, 7.1Hz, OCH₂), 4.19 (m, 2H, ArOCH₂), 4.00 (m, 1H, 0.5×OCH₂), 3.80 (m, 3H,0.5×OCH₂+OCH₂), 1.40 (t, 3H, J=7.1 Hz, CH₃).

3-[2-Azido-2-(2-hydroxyethoxy)ethoxy]benzoic acid

3-[2-Azido-2-(2-hydroxyethoxy)ethoxy]benzoic acid ethyl ester (2.39 g,8.10 mmol) was dissolved in ethanol (22 mL) and 4 M sodium hydroxidesolution (22 mL) was added. The mixture was stirred at room temperaturefor 3.5 h and the volume was reduced by ¾ under vacuum. The resultingmixture was diluted with water (50 mL) and acidified to pH 1-2 with 2 Mhydrochloric acid. This mixture was extracted with dichloromethane (3×80mL). The combined organic phases were washed with saturated brine (150mL), dried (MgSO₄) and concentrated under reduced pressure to afford3-[2-azido-2-(2-hydroxyethoxy)ethoxy]benzoic acid (2.20 g, 99%) as acolorless oil, which was used without further purification.

¹H NMR (300 MHz, CDCl₃) δ 7.76 (dt, 1H, J=7.7, 1.2 Hz, ArH), 7.65 (dd,1H, J=2.4, 1.4 Hz, ArH), 7.41 (t, 1H, J=8.0 Hz, ArH), 7.19 (ddd, 1H,J=8.2, 2.7, 1.0 Hz, ArH), 4.90 (t, 1H, J=5.1 Hz, CH), 4.21 (m, 2H,ArOCH₂), 4.03 (m, 1H, 0.5×OCH₂), 3.81 (m, 3H, 0.5×OCH₂+OCH₂).

3-[2-Azido-2-(2-ethoxycarbonylmethoxyethoxy)ethoxy]benzoic acid

To an ice-cold solution of 3-[2-azido-2-(2-hydroxyethoxy)ethoxy]benzoicacid (2.20 g, 8.31 mmol) in anhydrous THF (25 mL) was added sodiumhydride (0.99 g, 24.9 mmol) and the mixture was stirred at 0° C. for 10min. Ethyl bromoacetate (3.05 g, 2.03 mL, 18.3 mmol) was added and thereaction mixture was allowed to warm up to room temperature over 5 h.The reaction was quenched with ice-water (20 mL) and washed withdichloromethane (2×100 mL). The aqueous layer was acidified to pH 1-2 bythe addition of 2 M hydrochloric acid and was extracted withdichloromethane (3×50 mL). The combined organic layers were dried(MgSO₄) and concentrated under reduced pressure to afford a colorlessoil. This crude material was purified using a Biotage Isolera automatedchromatography system under normal phase conditions (silica column,gradient of 0→60% ethyl acetate in dichloromethane) with detection at254 nm to give3-[2-azido-2-(2-ethoxycarbonylmethoxyethoxy)ethoxy]benzoic acid (1.27 g,43%), as a colorless oil.

R_(f) 0.6 (ethyl acetate-dichloromethane, 4:6 v/v).

¹H NMR (300 MHz, CDCl₃) δ 7.75 (dt, 1H, J=7.7, 1.2 Hz, ArH), 7.64 (dd,1H, J=2.7, 1.5 Hz, ArH), 7.40 (t, 1H, J=8.0 Hz, ArH), 7.19 (ddd, 1H,J=8.3, 2.7, 1.0 Hz, ArH), 4.95 (dd, 1H, J=5.5, 4.6 Hz, CH), 4.14 (m, 7H,ArOCH₂+0.5×OCH₂+OCH₂+OCH₂C(O)), 3.86 (m, 3H, 0.5×OCH₂+OCH₂), 1.28 (t,3H, J=7.2 Hz, CH₃).

N-(2-Aminoethyl)-2,2,2-trifluoroacetamide Trifluoroacetate

To an ice-cold solution of N-Boc-ethylenediamine (2.08 g, 2.05 mL, 13.0mmol) in anhydrous THF (8 mL) was slowly added ethyl trifluoroacetate(1.85 g, 1.55 mL, 13.0 mmol) and the reaction mixture was stirred atroom temperature overnight. The resulting solution was concentratedunder reduced pressure to give tert-butyl[2-(2,2,2-trifluoroacetamido)ethyl]carbamate (3.30 g, 99%), as a whitesolid which was used without further purification.

tert-Butyl [2-(2,2,2-trifluoroacetamido)ethyl]carbamate (1.04 g, 4.06mmol) was dissolved in trifluoroacetic acid (5 mL) and stirred at roomtemperature for 30 min. The resulting mixture was concentrated underreduced pressure and the residual trifluoroacetic acid wasazeotropically removed with chloroform (3×10 mL). The material obtainedwas dried in vacuo at 50° C. for 2 h to giveN-(2-aminoethyl)-2,2,2-trifluoroacetamide trifluoroacetate (1.07 g,99%), as a yellow oil which was used without further purification.

¹H NMR (300 MHz, d₆-DMSO) δ 9.58 (br t, 1H, NH), 7.99 (br s, 3H, NH₃ ⁺),3.43 (m, 2H, CH₂N), 2.97 (m, 2H, CH₂N).

[2-(1-Azido-2-{3-[2-(2,2,2-trifluoroacetamido)ethylcarbamoyl]phenoxy}ethoxy)ethoxy]aceticacid ethyl ester

To a solution of N-(2-aminoethyl)-2,2,2-trifluoroacetamidetrifluoroacetate (0.79 g, 2.94 mmol) in anhydrous DMF (15 mL) was addedHunig's base (0.95 g, 1.28 mL, 7.34 mmol),3-[2-azido-2-(2-ethoxycarbonylmethoxyethoxy)ethoxy]benzoic acid (0.87 g,2.45 mmol) and PyBOP (1.41 g, 2.69 mmol). The reaction mixture wasstirred at room temperature overnight and was then quenched with 1 Mhydrochloric acid (20 mL) and extracted with ethyl acetate (3×30 mL).The organic layers were combined, dried (MgSO₄) and concentrated underreduced pressure to give an orange oil. This oil was purified using aBiotage Isolera automated chromatography system under normal phaseconditions (silica column, gradient of 0→90% ethyl acetate indichloromethane) with detection at 254 nm to give[2-(1-azido-2-{3-[2-(2,2,2-trifluoroacetamido)ethylcarbamoyl]phenoxy}ethoxy)ethoxy]aceticacid ethyl ester (1.16 g, 96%), as a colorless oil.

R_(f) 0.48 (dichloromethane-ethyl acetate, 1:1, v/v).

¹H NMR (300 MHz, CDCl₃) δ 8.01 (br, 1H, NH), 7.39 (m, 3H, 3×ArH), 7.10(dt, 1H, J=5.8, 3.0 Hz, ArH), 6.97 (br, 1H, NH), 4.93 (t, 1H, J=5.1 Hz,CH), 4.18 (m, 6H, ArOCH₂+OCH₂+OCH₂C(O)), 4.04 (m, 1H, 0.5×OCH₂), 3.87(m, 1H, 0.5×OCH₂), 3.81 (m, 2H, OCH₂), 3.69 (m, 2H, CH₂N), 3.62 (m, 2H,CH₂N), 1.30 (t, 3H, J=7.1 Hz, CH₃).

[2-(1-Azido-2-{3-[2-(tert-butoxycarbonyl)ethylcarbamoyl]phenoxy}ethoxy)ethoxy]aceticacid ethyl ester

To a solution of3-[2-azido-2-(2-ethoxycarbonylmethoxyethoxy)ethoxy]benzoic acid (0.82 g,2.33 mmol) in ethyl acetate (25 mL) was added N-Boc-ethylenediamine (410mg, 0.41 mL, 2.56 mmol), PyBOP (1.34 g, 2.56 mmol) and Hunig's base(0.66 g, 0.41 mL, 5.12 mmol). The resulting reaction mixture was stirredat room temperature overnight. The solution was partitioned betweenethyl acetate (20 mL) and 1 M hydrochloric acid (20 mL). The aqueousphase was separated and extracted with ethyl acetate (2×20 mL). Theorganic phases were combined and washed with saturated brine (50 mL),dried (MgSO4) and concentrated under reduced pressure to give a yellowoil. This oil was purified using a Biotage Isolera automatedchromatography system under normal phase conditions (silica column,gradient of 0→80% ethyl acetate in dichloromethane) with detection at254 nm. The fractions containing the product were combined andevaporated under reduced pressure. The material obtained was dissolvedin ethyl acetate (50 mL) and washed with saturated aqueous sodiumbicarbonate solution (50 mL), water (50 mL) and saturated brine (50 mL)before being dried (MgSO4) and concentrated under reduced pressure togive[2-(1-azido-2-{3-[2-(tert-butoxycarbonyl)ethylcarbamoyl]phenoxy}ethoxy)ethoxy]aceticacid ethyl ester (0.98 g, 86%) as a yellow oil.

R_(f) 0.41 (dichloromethane-ethyl acetate, 1:1, v/v).

¹H NMR (300 MHz, CDCl₃) δ 7.42 (m, 2H, 2×ArH), 7.34 (t, 1H, J=7.8 Hz,ArH), 7.07 (m, 1H, ArH), 5.04 (br, 1H, NH), 4.94 (t, 1H, J=5.1 Hz, CH),4.22 (m, 2H, OCH₂), 4.15 (m, 4H, ArOCH₂+OCH₂C(O)), 4.04 (m, 1H,0.5×OCH₂), 3.88 (m, 1H, 0.5×OCH₂), 3.81 (m, 2H, OCH₂), 3.57 (m, 2H,CH₂N), 3.41 (m, 2H, CH₂N), 1.44 (s, 9H, 3×CH₃), 1.30 (t, 3H, J=7.1 Hz,CH₃).

Sodium(2-{2-[3-(2-aminoethylcarbamoyl)phenoxy]-1-azidoethoxy}ethoxy)acetate

Method A

[2-(1-Azido-2-{3-[2-(2,2,2-trifluoroacetamido)ethylcarbamoyl]phenoxy}ethoxy)ethoxy]aceticacid ethyl ester (1.16 g, 2.36 mmol) was dissolved in ethanol (7 mL) and4 M sodium hydroxide solution (7 mL) was added. The resulting mixturewas stirred at room temperature for 2.5 h before being concentratedunder reduced pressure. The residue was dissolved in water (80 mL) andwashed with dichloromethane (2×70 mL). The aqueous layer was acidifiedto pH 2 using 1 M hydrochloric acid and washed with dichloromethane(3×70 mL). The resulting aqueous layer was neutralised to pH 8 using 1 Msodium hydroxide solution and evaporated under reduced pressure to givea white solid which was entrained with dichloromethane and methanol(2×140 mL, 1:1, v/v). The solid obtained was collected by suctionfiltration and discarded. The filtrate was concentrated under reducedpressure to give a residual crude gum. This gum was entrained withdichloromethane and methanol (10 mL, 9:1, v/v) and the insoluble whitesolid obtained was collected by suction filtration and discarded. Thefiltrate was concentrated under reduced pressure to give sodium(2-{2-[3-(2-aminoethylcarbamoyl)phenoxy]-1-azidoethoxy}ethoxy)acetate(1.11 g, 100%), as a white glassy foam.

Method B

To a solution of[2-(1-azido-2-{3-[2-(tert-butoxycarbonyl)ethylcarbamoyl]phenoxy}ethoxy)ethoxy]aceticacid ethyl ester (0.59 g, 1.20 mmol) in ethanol (4 mL) was added 4 Msodium hydroxide solution (4 mL) and the mixture was stirred for 2.5 h.The solution was concentrated under reduced pressure and the residue wasdissolved in water (15 mL). This aqueous mixture was acidified to pH 1-2using 2 M hydrochloric acid and was extracted with dichloromethane (3×20mL). The combined organic layers were dried (MgSO₄) and thenconcentrated under reduced pressure to give[2-(1-azido-2-{3-[2-(tert-butoxycarbonyl)ethylcarbamoyl]phenoxy}ethoxy)ethoxy]aceticacid (0.52 g, 92%), as a colorless oil.

To a solution of[2-(1-azido-2-{3-[2-(tert-butoxycarbonyl)ethylcarbamoyl]phenoxy}ethoxy)ethoxy]aceticacid (50 mg, 0.11 mmol) in dichloromethane (1.5 mL) was addedtrifluoroacetic acid (80 μL) and the mixture was stirred at roomtemperature for 40 min. The solution was concentrated under reducedpressure to afford a colorless oil. This oil was partitioned betweendichloromethane (2 mL) and 1 M hydrochloric acid (2 mL). The organiclayer was discarded and the aqueous layer was adjusted to pH 8 using 3 Msodium hydroxide solution before being concentrated under reducedpressure. The white solid obtained was entrained with a mixture ofdichloromethane and methanol (2×10 mL, 1:1, v/v) and the precipitate wasdiscarded. The filtrate was evaporated and the residue was entrainedwith a mixture of dichloromethane and methanol (10 mL, 9:1, v/v) and theprecipitate was discarded. The filtrate was concentrated under reducedpressure to give sodium(2-{2-[3-(2-aminoethylcarbamoyl)phenoxy]-1-azidoethoxy}ethoxy)acetate(43 mg, 99%), as a white glassy solid.

¹H NMR (300 MHz, D₂O) δ 7.31 (m, 3H, 3×ArH), 7.11 (ddd, 1H, J=7.8, 2.6,1.5 Hz, ArH), 4.99 (t, 1H, J=4.5 Hz, CH), 4.15 (m, 2H, ArOCH₂), 3.94 (m,1H, 0.5×OCH₂), 3.79 (m, 3H, 0.5×OCH₂+OCH₂C(O)), 3.58 (m, 4H, OCH₂+CH₂N),3.10 (t, 2H, J=5.8 Hz CH₂N), 1.30 (t, 3H, J=7.1 Hz, CH₃).

2-(2-{1-Azido-2-[3-({2-[3,5-bis(methoxycarbonyl)benzamido]ethyl}carbamoyl)phenoxy]ethoxy}ethoxy)aceticacid (Fragment 2)

Trimethyl 1,3,5-benzenetricarboxylate (2.00 g, 7.93 mmol) was suspendedin methanol (180 mL) and 1 M sodium hydroxide solution (7.14 mL) wasadded. The mixture was stirred at room temperature for 18 h. Theresulting solution was concentrated under reduced pressure to afford awhite solid which was partitioned between dichloromethane (150 mL) andsaturated aqueous sodium bicarbonate solution (150 mL). The organicphase was separated and was extracted with saturated aqueous sodiumbicarbonate solution (150 mL) before being discarded. The combinedaqueous layers were acidified to pH 1-2 using concentrated hydrochloricacid and were extracted with ethyl acetate (2×150 mL). The combinedorganic layers were dried (MgSO₄) and concentrated to give3,5-bis(methoxycarbonyl)benzoic acid (1.66 g, 88%), as a white solid.

3,5-bis(Methoxycarbonyl)benzoic acid (0.80 g, 3.36 mmol) was dissolvedin ethyl acetate (20 mL) and N-hydroxysuccinimide (425 mg, 3.69 mmol)was added followed by N,N′-dicyclohexylcarbodiimide (0.76 g, 3.69 mmol).The resulting mixture was stirred at room temperature for 20 h. Thesuspension was filtered over Celite and the filtrate was washed withsaturated aqueous sodium bicarbonate solution (2×100 mL), water (100 mL)and saturated brine (100 mL). The organic layer was dried (MgSO₄) andconcentrated under reduced pressure to afford a white glassy solid. Thismaterial was purified using a Biotage Isolera automated chromatographysystem under normal phase conditions (silica column, gradient of 0→100%ethyl acetate in petrol) with detection at 254 nm to giveN-hydroxysuccinimide 3,5-bis(methoxycarbonyl)benzoate (1.06 g, 95%), asa white solid.

R_(f) 0.44 (petrol-ethyl acetate, 4:6, v/v).

To a solution of N-hydroxysuccinimide 3,5-bis(methoxycarbonyl)benzoate(0.95 g, 2.83 mmol) and sodium(2-{2-[3-(2-aminoethylcarbamoyl)phenoxy]-1-azidoethoxy}ethoxy)acetate(0.92 g, 2.36 mmol) in anhydrous DMF (15 mL) was added Hunig's base(0.61 g, 0.83 mL, 4.72 mmol) and the mixture was stirred at roomtemperature overnight. The resulting mixture was diluted with ethylacetate (20 mL) and quenched with 1 M hydrochloric acid (20 mL). Theaqueous phase was separated and was extracted with ethyl acetate (3×30mL) and the combined organic phases were washed with water (5×50 mL) andsaturated brine (50 mL). The resulting solution was dried (MgSO₄) beforebeing concentrated under reduced pressure to give a white glassy solid.This material was purified using a Biotage Isolera automatedchromatography system under normal phase conditions (silica column,gradient of 0→50% methanol in dichloromethane) with detection at 254 nmto give2-(2-{1-azido-2-[3-({2-[3,5-bis(methoxycarbonyl)benzamido]ethyl}carbamoyl)phenoxy]ethoxy}ethoxy)aceticacid (Fragment 2) (0.92 g, 66%), as a white glassy solid.

R_(f) 0.37 (dichloromethane-methanol, 83:17, v/v).

¹H NMR (300 MHz, d₆-DMSO) δ 9.42 (br s, 1H, NH), 8.99 (br s, 1H, NH),8.66 (d, 2H, J=1.5 Hz, 2×ArH), 8.53 (t, 1H, J=1.5 Hz, ArH), 7.50 (m, 1H,ArH), 7.43 (d, 1H, J=7.7 Hz, ArH), 7.33 (t, 1H, J=7.9 Hz, ArH), 7.07(dd, 1H, J=8.0, 2.4 Hz, ArH), 5.21 (t, 1H, J=5.0 Hz, CH), 4.24 (dd, 1H,J=10.6, 4.4 Hz, 0.5×ArOCH₂), 4.07 (dd, 1H, J=10.6, 5.9 Hz, 0.5×ArOCH₂),3.88 (m, 8H, OCH₂+2×OCH₃), 3.84 (m, 1H, 0.5×OCH₂), 3.77 (m, 1H,0.5×OCH₂), 3.62 (m, 2H, OCH₂C(O)), 3.40 (m, 4H, 2×CH₂N).

Pre-Cleavable SCTTP

3′-O-Pivaloyl-5-(3-aminopropynyl)-2′-deoxyuridine trifluoroacetate (1.04g, 2.16 mmol) was dissolved in anhydrous DMF (20 mL). Hunig's base (0.80g, 1.08 mL, 6.18 mmol) followed by2-(2-{1-azido-2-[3-({2-[3,5-bis(methoxycarbonyl)benzamido]ethyl}carbamoyl)phenoxy]ethoxy}ethoxy)aceticacid (0.91 g, 1.55 mmol) and HBTU (0.62 g, 1.62 mmol) were then added.The reaction mixture was stirred at room temperature for 20 h. and wasquenched with 1 M hydrochloric acid (20 mL) and diluted with ethylacetate (20 mL). The aqueous phase was separated and extracted withethyl acetate (2×50 mL). The combined organic phases were washed withwater (5×60 mL), saturated aqueous sodium bicarbonate solution (60 mL)and brine (60 mL), before being dried (MgSO₄) and concentrated underreduced pressure to give an off-white solid. This solid was purifiedusing a Biotage Isolera automated chromatography system under normalphase conditions (silica column, gradient of 0→10% methanol indichloromethane) with detection at 254 nm to afford Pre-Cleavable SCTTP(1.20 g, 83%), as an off-white solid.

R_(f) 0.42 (dichloromethane-methanol, 92:8, v/v).

¹H NMR (300 MHz, d₆-DMSO) δ 11.69 (s, 1H, deoxyuridine NH), 9.09 (br t,1H, NH), 8.68 (d, 2H, J=1.6 Hz, 2×ArH), 8.64 (br t, 1H, NH), 8.58 (t,1H, J=1.6 Hz, ArH), 8.26 (t, 1H, J=5.8 Hz, NH), 8.16 (s, 1H,deoxyuridine CH), 7.46 (m, 2H, 2×ArH), 7.38 (t, 1H, J=7.8 Hz, ArH), 7.13(m, 1H, ArH), 6.12 (dd, 1H, J=8.3, 6.0 Hz, CH), 5.27 (m, 1H, CHN), 5.16(m, 2H, CHOPiv+OH), 4.23 (dd, 1H, J=10.4, 4.5 Hz, 0.5×ArOCH₂), 4.13 (m,2H, 0.5×ArOCH₂+0.5×OCH₂), 3.88 (m, 10H, CH₂N+OCH₂+2×OCH₃), 3.85 (m, 1H,0.5×OCH₂), 3.68 (m, 2H, deoxyuridine CH₂), 3.63 (m, 2H, OCH₂C(O)), 3.46(m, 4H, 2×CH₂N), 2.27 (m, 2H, deoxyuridine CH₂), 1.15 (s, 9H, 3×CH₃).

Penta-Triethylammonium Cleavable SCTTP

Pre-Cleavable SCTTP (390 mg, 0.42 mmol) was dissolved in 1,4-dioxane(0.39 mL) and pyridine (1.2 mL) and the flask was evacuated and purgedwith a nitrogen atmosphere. 1.0 M Salicyl chlorophosphite solution in1,4-dioxane (460 μL, L, 0.46 mmol) was added and the reaction mixturewas stirred for 10 min. 0.5 M Tributylammonium pyrophosphate solution inDMF (1.25 mL, 0.63 mmol) and tributylamine (358 mg, 460 μL, 1.93 mmol)were added and the reaction mixture was stirred for 20 min. 1% Iodinesolution in pyridine and water (8.4 mL, 98:2) was added and the solutionwas stirred for 15 min before being quenched with 5% aqueous sodiumthiosulfate solution (1 mL) and concentrated under reduced pressure. Thematerial obtained was purified using a Biotage Isolera automatedchromatography system under reversed-phase conditions (C₁₈ column,gradient of 0→40% acetonitrile in 0.1 M TEAA at pH 7.0) with detectionat 287 nm to afford, after freeze-drying, impure protected CleavableSCTTP (360 mg) which was used without further purification.

Protected Cleavable SCTTP (360 mg) was dissolved in water (3.0 mL) andthe resulting solution was stirred for 15 min before addition of 1 Msodium hydroxide solution (3.0 mL). The mixture was stirred at roomtemperature for 2 h and was quenched with 1 M aqueous triethylammoniumbicarbonate (TEAB) solution (pH 8.5, 10 mL). The resulting solution waslyophilized overnight and the residue was dissolved in water (4 mL) togive a concentration of 100 mg/mL. This solution was purified bysemi-preparative HPLC injecting 100 μL portions and collecting theeluent containing the pure substance. The combined fractions werereduced in volume by removing the acetonitrile and most of the water andfinally lyophilized to give penta-triethylammonium Cleavable SCTTP (233mg, 35 over two steps), as a white solid.

HPLC Conditions

Column: Phenomenex Luna C18(2), 15 mm×250 mm

Solvent Gradient: 90% 0.1 M aqueous TEAB to 84.5% 0.1 M aqueous TEABover 22 min with the balance being acetonitrile.

Flow Rate: 7.8 mL/min

Temp: 30° C.

Detection: UV at 287 nm

Under these conditions the product had a retention time of ca. 17-19min.

Example 5 Synthesis of Supercharged CTP (SCCTP)5′-O-Dimethoxytrityl-5-iodo-2′-deoxycytidine

5-Iodo-2′-deoxycytidine (500 mg, 1.42 mmol) was dissolved in anhydrouspyridine (6.0 mL) and stirred under nitrogen. After 5 min,4-(dimethylamino)pyridine (10 mg, 0.08 mmol) and triethylamine (22 mg,30 μL, 0.21 mmol) were added to the reaction mixture, followed by4,4′-dimethoxytriphenylmethyl chloride (576 mg, 1.70 mmol). Theresulting mixture was stirred overnight at room temperature and quenchedwith methanol (˜1.0 mL) before being concentrated under reducedpressure. The residue obtained was partitioned between a saturatedaqueous sodium bicarbonate solution (25 mL) and dichloromethane (25 mL).The layers were separated and the aqueous phase was extracted withdichloromethane (25 mL). The organic phases were combined, dried (MgSO₄)and concentrated under reduced pressure to give a yellow semi-solid.This crude material was purified using a Biotage Isolera automatedchromatography system under normal phase conditions (silica column,gradient of 1.5→24% methanol in dichloromethane) with detection at 254nm to give 5′-O-dimethoxytrityl-5-iodo-2′-deoxycytidine (0.58 g, 62%),as a white solid.

R_(f) 0.35 (dichloromethane-methanol, 94:6, v/v).

¹H NMR (300 MHz, d₆-DMSO): 7.97 (s, 1H, CH), 7.89 (br, 1H, 0.5×NH₂),7.32 (m, 9H, 9×ArH), 6.91 (m, 4H, 4×ArH), 6.66 (br, 1H, 0.5×NH₂), 6.11(t, J=6.7 Hz, 1H, CH), 5.29 (d, J=4.1 Hz, 1H, OH), 4.20 (m, 1H, CH),3.91 (m, 1H, CH), 3.74 (s, 6H, 2×OCH₃), 3.17 (m, 2H, CH₂), 2.21 (m, 1H,0.5×CH₂), 2.09 (m, 1H, 0.5×CH₂).

4-N-3′-O-Di-pivaloyl-5′-O-dimethoxytrityl-5-iodo-2′-deoxycytidine

5′-O-Dimethoxytrityl-5-iodo-2′-deoxycytidine (0.57 g, 0.87 mmol) wasdissolved in anhydrous acetonitrile (11.4 mL). 4-(Dimethylamino)pyridine(21 mg, 0.17 mmol) and triethylamine (369 mg, 0.51 mL, 3.65 mmol)followed by trimethylacetic anhydride (0.65 g, 0.71 mL, 3.48 mmol) wereadded. The reaction mixture was stirred and heated at reflux for 20 hunder nitrogen, then allowed to cool to room temperature and methanol(˜1.0 mL) was added. The resulting solution was concentrated underreduced pressure to give a crude residue which was purified using aBiotage Isolera automated chromatography system under normal phaseconditions (silica column, gradient of 3→30% ethyl acetate in petrol)with detection at 254 nm to give4-N-3′-O-di-pivaloyl-5′-O-dimethoxytrityl-5-iodo-2′-deoxycytidine (0.70g, 97%), as a colorless oil which solidified on standing.

R_(f) 0.32 (petrol-ethyl acetate, 85:15, v/v).

¹H NMR (300 MHz, d₆-DMSO): 7.32 (m, 9H, 9×ArH), 6.90 (m, 4H, 4×ArH),6.08 (t, J=6.8 Hz, 1H, CH), 5.21 (m, 1H, CH), 4.04 (m, 1H, CH), 3.74 (s,6H, 2×OCH₃), 3.30 (obscured, 2H, CH₂), 2.40 (obscured, 2H, CH₂), 1.19(s, 9H, 3×CH₃), 1.13 (s, 9H, 3×CH₃).

3′-O-Pivaloyl-5′-O-dimethoxytrityl-5-iodo-2′-deoxycytidine

4-N-3′-O-Di-pivaloyl-5′-O-dimethoxytrityl-5-iodo-2′-deoxycytidine (0.70g, 0.85 mmol) was dissolved in anhydrous methanol (8.8 mL) and Hunig'sbase (165 mg, 221 μL, 1.28 mmol) was added. The reaction mixture washeated in a sealed vial at ˜110° C. for 3 h before being concentratedunder reduced pressure. The resulting impure material was purified usinga Biotage Isolera automated chromatography system under normal phaseconditions (silica column, gradient of 1→10% methanol indichloromethane) with detection at 254 nm to give3′-O-pivaloyl-5′-O-dimethoxytrityl-5-iodo-2′-deoxycytidine (0.50 g,80%), as a white solid.

R_(f) 0.33 (dichloromethane-methanol, 96:4, v/v).

¹H NMR (300 MHz, d₆-DMSO): δ 8.02 (s, 1H, CH), 7.94 (br, 1H, 0.5×NH₂),7.33 (m, 9H, 9×ArH), 6.90 (m, 4H, 4×ArH), 6.73 (br, 1H, 0.5×NH₂), 6.12(t, J=7.0 Hz, 1H, CH), 5.19 (m, 1H, CH), 3.98 (m, 1H, CH), 3.74 (s, 6H,2×OCH₃), 3.27 (m, 2H, CH₂), 2.33 (m, 2H, CH₂), 1.13 (s, 9H, 3×CH₃).

3′-O-Pivaloyl-5′-O-dimethoxytrityl-5-(N-Boc-3-amido-propynyl)-2′-deoxycytidine

3′-O-Pivaloyl-5′-O-dimethoxytrityl-5-iodo-2′-deoxycytidine (0.50 g, 0.68mmol), copper(I) iodide (26 mg, 0.14 mmol) andtetrakis(triphenylphosphine)palladium(0) (81 mg, 0.07 mmol) weredissolved in ethyl acetate (5.9 mL) and the flask was evacuated thenpurged with nitrogen four times. Hunig's base (175 mg, 230 μL, 1.35mmol) and N-Boc-propargylamine (315 mg, 2.03 mmol) were added and theresulting mixture was evacuated and purged with nitrogen before beingleft to stir at room temperature overnight. The reaction mixture wasdiluted with ethyl acetate (25 mL) and washed with 5% aqueous EDTAsolution (25 mL). The layers were separated and the aqueous phase waswashed with ethyl acetate (10 mL). The combined organic layers werewashed with saturated brine (25 mL), dried (MgSO₄) and concentratedunder reduced pressure to give an orange solid. This solid was purifiedusing a Biotage Isolera automated chromatography system under normalphase conditions (silica column, gradient of 1→13% methanol indichloromethane) with detection at 254 nm to give3′-O-pivaloyl-5′-O-dimethoxytrityl-5-(N-Boc-3-amido-propynyl)-2′-deoxycytidine(470 mg, 91%), as a pale yellow solid.

R_(f) 0.33 (dichloromethane-methanol, 96:4, v/v).

¹H NMR (300 MHz, d₆-DMSO): δ 7.94 (s, 1H, CH), 7.92 (br, 1H, 0.5×NH₂),7.31 (m, 9H, 9×ArH), 6.89 (m, 5H, 4×ArH+0.5×NH₂), 6.15 (t, J=7.0 Hz, 1H,CH), 5.23 (m, 1H, CH), 4.02 (m, 1H, CH), 3.85 (m, 2H, CH₂), 3.74 (s, 6H,2×OCH₃), 3.38 (obscured, 1H, 0.5×CH₂), 3.17 (m, 1H, 0.5×CH₂), 2.35 (m,2H, CH₂), 1.38 (s, 9H, 3×CH₃), 1.13 (s, 9H, 3×CH₃).

3′-O-Pivaloyl-5-(3-aminopropynyl)-2′-deoxycytidine Trifluoroacetate

3′-O-Pivaloyl-5′-O-dimethoxytrityl-5-(N-Boc-3-amido-propynyl)-2′-deoxycytidine(465 mg, 0.61 mmol) was dissolved in anhydrous dichloromethane (11.9 mL)and trifluoroacetic acid (2.77 g, 1.87 mL, 24.3 mmol) was addeddropwise. The mixture was stirred for 2 h at room temperature and wassubsequently diluted with dichloromethane (20 mL). The resultingsolution was concentrated under reduced pressure and the residualtrifluoroacetic acid was azeotropically removed with dichloromethane(3×20 mL). The impure material was purified using a Biotage Isoleraautomated chromatography system under reversed-phase conditions (C₁₈column, gradient of 0→40% acetonitrile in 0.1% aqueous trifluoroaceticacid) with detection at 254 nm to afford, after freeze-drying,3′-O-pivaloyl-5-(3-aminopropynyl)-2′-deoxycytidine trifluoroacetate(Fragment 1) (269 mg, 92%), as an off-white solid.

¹H NMR (300 MHz, DMSO-d₆): δ 8.33 (br, 1H, 0.5×NH₂), 8.27 (s, 1H, CH),8.24 (br, 3H, NH₃ ⁺), 7.32 (br, 1H, 0.5×NH₂), 6.14 (m, 1H, CH), 5.19 (m,1H, CH), 3.98 (m, 3H, CH+CH₂), 3.64 (m, 2H, CH₂), 2.28 (m, 2H, CH₂),1.17 (s, 9H, 3×CH₃).

3-[(1,3)Dioxolan-2-ylmethoxy]benzoic acid ethyl ester

Ethyl 3-hydroxybenzoate (3.50 g, 21.1 mmol), bromoethyl-1,3-dioxolane(14.1 g, 8.72 mL, 84.3 mmol), potassium carbonate (5.83 g, 42.1 mmol)and sodium iodide (1.26 g, 8.43 mmol) were dissolved in anhydrous DMF(10 mL) and the reaction mixture was stirred at 120° C. for 20 h. Thesuspension was cooled to room temperature and quenched with water (30mL). The aqueous phase was extracted with ethyl acetate (3×50 mL). Theorganic layers were combined, washed with water (5×100 mL) and saturatedbrine (100 mL), before being dried (MgSO₄) and concentrated underreduced pressure to afford a residual orange oil. This oil was purifiedusing a Biotage Isolera automated chromatography system under normalphase conditions (silica column, gradient of 0→30% ethyl acetate inpetrol) with detection at 254 nm to give3-[(1,3)dioxolan-2-ylmethoxy]benzoic acid ethyl ester (5.09 g, 96%), asa colorless oil.

R_(f) 0.40 (petrol-ethyl acetate, 75:25, v/v).

¹H NMR (400 MHz, CDCl₃): δ 7.66 (dt, J=7.5, 1.1 Hz, 1H, ArH), 7.59 (dd,J=2.6, 1.5 Hz, 1H, ArH), 7.34 (t, J=7.9 Hz, 1H, ArH), 7.14 (ddd, J=8.3,2.6, 1.0 Hz, 1H, ArH), 5.31 (t, J=4.1 Hz, 1H, CH), 4.36 (q, J=7.2 Hz,2H, OCH₂), 4.02 (m, 6H, OCH₂CH₂O+ArOCH₂), 1.39 (t, J=7.1 Hz, 3H, CH₃).

3-[2-Azido-2-(2-hydroxyethoxy)ethoxy]benzoic acid ethyl ester

To a mixture of 3-[(1,3)dioxolan-2-ylmethoxy]benzoic acid ethyl ester(5.08 g, 20.1 mmol) and azidotrimethylsilane (2.55 g, 2.94 mL, 22.2mmol) was added tin(IV) chloride (336 mg, 151 μL, 1.29 mmol). Thereaction mixture was stirred at room temperature for 2 h. The resultingmixture was diluted with 2% aqueous methanol (60 mL) and stirred for 30min before being concentrated under reduced pressure. The residue wasazeotropically dried with ethanol (2×30 mL) to afford a colorless oil.This oil was purified using a Biotage Isolera automated chromatographysystem under normal phase conditions (silica column, gradient of 0→70%ethyl acetate in petrol) with detection at 254 nm to give3-[2-azido-2-(2-hydroxyethoxy)ethoxy]benzoic acid ethyl ester (3.59 g,59%), as a colorless oil.

R_(f) 0.41 (petrol-ethyl acetate, 53:47, v/v).

¹H NMR (300 MHz, CDCl₃): δ 7.69 (dt, J=7.7, 1.2 Hz, 1H, ArH), 7.59 (dd,J=2.6, 1.5 Hz, 1H, ArH), 7.36 (t, J=8.0 Hz, 1H, ArH), 7.13 (ddd, J=8.3,2.7, 1.0 Hz, 1H, ArH), 4.89 (t, J=5.1 Hz, 1H, CH), 4.38 (q, J=7.1 Hz,2H, OCH₂), 4.19 (m, 2H, ArOCH₂), 4.00 (m, 1H, 0.5×OCH₂), 3.80 (m, 3H,0.5×OCH₂+OCH₂), 1.40 (t, J=7.1 Hz, 3H, CH₃).

3-[2-Azido-2-(2-hydroxyethoxy)ethoxy]benzoic acid

3-[2-Azido-2-(2-hydroxyethoxy)ethoxy]benzoic acid ethyl ester (3.51 g,11.9 mmol) was dissolved in ethanol (30 mL) and 4 M aqueous sodiumhydroxide (30 mL) was added. The mixture was stirred at room temperaturefor 3.5 h and the volume was then reduced by ¾ under vacuum. Theresulting mixture was diluted with water (50 mL) and acidified to pH 1-2with 2 M hydrochloric acid. This mixture was extracted withdichloromethane (3×100 mL). The combined organic phases were washed withsaturated brine (150 mL), dried (MgSO₄) and concentrated under reducedpressure to afford 3-[2-azido-2-(2-hydroxyethoxy)ethoxy]benzoic acid(3.39 g, quantitative), as a colorless oil which was used withoutfurther purification.

¹H NMR (300 MHz, CDCl₃): δ 7.76 (dt, J=7.7, 1.2 Hz, 1H, ArH), 7.65 (dd,J=2.4, 1.4 Hz, 1H, ArH), 7.41 (t, J=8.0 Hz, 1H, ArH), 7.19 (ddd, J=8.2,2.7, 1.0 Hz, 1H, ArH), 4.90 (t, J=5.1 Hz, 1H, CH), 4.21 (m, 2H, ArOCH₂),4.03 (m, 1H, 0.5×OCH₂), 3.81 (m, 3H, 0.5×OCH₂+OCH₂).

3-[2-Azido-2-(2-ethoxycarbonylmethoxyethoxy)ethoxy]benzoic acid

To an ice-cold solution of 3-[2-azido-2-(2-hydroxyethoxy)ethoxy]benzoicacid (1.42 g, 5.31 mmol) in anhydrous THF (18 mL) was added sodiumhydride (0.64 g, 15.9 mmol) and the mixture was stirred at 0° C. for 10min. Ethyl bromoacetate (1.95 g, 1.30 mL, 11.7 mmol) was added and thereaction mixture was allowed to warm up to room temperature over 5 h.The resulting mixture was quenched with ice-water (20 mL) and washedwith dichloromethane (2×70 mL). The aqueous layer was acidified to pH1-2 using 2 M hydrochloric acid and was extracted with dichloromethane(3×70 mL). The combined organic layers were dried (MgSO₄) andconcentrated under reduced pressure to afford a colorless oil. Thismaterial was purified using a Biotage Isolera automated chromatographysystem under normal phase conditions (silica column, gradient of 0→60%ethyl acetate in dichloromethane) with detection at 254 nm to give3-[2-azido-2-(2-ethoxycarbonylmethoxyethoxy)ethoxy]benzoic acid (0.62 g,33%), as a colorless oil.

R_(f) 0.45 (ethyl acetate-dichloromethane, 4:6 v/v).

¹H NMR (300 MHz, CDCl₃): δ 7.75 (dt, J=7.7, 1.2 Hz, 1H, ArH), 7.64 (dd,J=2.7, 1.5 Hz, 1H, ArH), 7.40 (t, J=8.0 Hz, 1H, ArH), 7.19 (ddd, J=8.3,2.7, 1.0 Hz, 1H, ArH), 4.95 (dd, J=5.5, 4.6 Hz, 1H, CH), 4.14 (m, 7H,ArOCH₂+0.5×OCH₂+OCH₂+OCH₂C(O)), 3.86 (m, 3H, 0.5×OCH₂+OCH₂), 1.28 (t,J=7.2 Hz, 3H, CH₃).

N-(2-Aminoethyl)-2,2,2-trifluoroacetamide Trifluoroacetate

To an ice-cold solution of N-Boc-ethylenediamine (2.08 g, 2.05 mL, 13.0mmol) in anhydrous THF (8 mL) was slowly added ethyl trifluoroacetate(1.85 g, 1.55 mL, 13.0 mmol) and the reaction mixture was stirred atroom temperature overnight. The resulting solution was concentratedunder reduced pressure to give tert-butyl[2-(2,2,2-trifluoroacetamido)ethyl]carbamate (3.30 g, 99%), as a whitesolid which was used without further purification.

tert-Butyl [2-(2,2,2-trifluoroacetamido)ethyl]carbamate (1.04 g, 4.06mmol) was dissolved in trifluoroacetic acid (5 mL) and stirred at roomtemperature for 30 min. The resulting mixture was concentrated underreduced pressure and the residual trifluoroacetic acid wasazeotropically removed with chloroform (3×10 mL). The material obtainedwas dried in vacuo at 50° C. for 2 h to giveN-(2-aminoethyl)-2,2,2-trifluoroacetamide trifluoroacetate (1.07 g,99%), as a yellow oil which was used without further purification.

¹H NMR (300 MHz, DMSO-d₆): δ 9.58 (br, 1H, NH), 7.99 (br, 3H, NH₃ ⁺),3.43 (m, 2H, CH₂N), 2.97 (m, 2H, CH₂N).

[2-(1-Azido-2-{3-[2-(2,2,2-trifluoroacetamido)ethylcarbamoyl]phenoxy}ethoxy)ethoxy]aceticacid ethyl ester

To a solution of N-(2-aminoethyl)-2,2,2-trifluoroacetamidetrifluoroacetate (1.31 g, 4.86 mmol) in anhydrous DMF (25 mL) was addedHunig's base (1.57 g, 2.12 mL, 12.1 mmol),3-[2-azido-2-(2-ethoxycarbonylmethoxyethoxy)ethoxy]benzoic acid (1.43 g,4.05 mmol) and PyBOP (2.32 g, 4.45 mmol). The reaction mixture wasstirred at room temperature for 20 h before being diluted with ethylacetate (20 mL) and quenched with 1 M hydrochloric acid (40 mL). Theaqueous phase was extracted with ethyl acetate (3×50 mL). The combinedorganic layers were washed with water (5×100 mL), saturated brine (100mL), dried (MgSO₄) and concentrated under reduced pressure to give aresidual yellow oil. This oil was purified using a Biotage Isoleraautomated chromatography system under normal phase conditions (silicacolumn, gradient of 0→90% ethyl acetate in dichloromethane) withdetection at 254 nm to give[2-(1-azido-2-{3-[2-(2,2,2-trifluoroacetamido)ethylcarbamoyl]phenoxy}ethoxy)ethoxy]aceticacid ethyl ester (1.75 g, 88%), as a colorless oil.

R_(f) 0.48 (dichloromethane-ethyl acetate, 1:1, v/v).

¹H NMR (300 MHz, CDCl₃): δ 8.01 (br, 1H, NH), 7.39 (m, 3H, 3×ArH), 7.10(dt, J=5.8, 3.0 Hz, 1H, ArH), 6.97 (br, 1H, NH), 4.93 (t, J=5.1 Hz, 1H,CH), 4.18 (m, 6H, ArOCH₂+OCH₂+OCH₂C(O)), 4.04 (m, 1H, 0.5×OCH₂), 3.87(m, 1H, 0.5×OCH₂), 3.81 (m, 2H, OCH₂), 3.69 (m, 2H, CH₂N), 3.62 (m, 2H,CH₂N), 1.30 (t, J=7.1 Hz, 3H, CH₃).

Sodium(2-{2-[3-(2-aminoethylcarbamoyl)phenoxy]-1-azidoethoxy}ethoxy)acetate

[2-(1-Azido-2-{3-[2-(2,2,2-trifluoroacetamido)ethylcarbamoyl]phenoxy}ethoxy)ethoxy]aceticacid ethyl ester (0.90 g, 1.83 mmol) was dissolved in ethanol (7 mL) and4 M aqueous sodium hydroxide (7 mL) was added. The resulting mixture wasstirred at room temperature for 2.5 h before being concentrated underreduced pressure. The material obtained was dissolved in water (55 mL)and washed with dichloromethane (2×50 mL). The aqueous layer wasacidified to pH 2 using 1 M hydrochloric acid and washed withdichloromethane (2×50 mL). The organic layer was removed and discardedwhile the aqueous layer was neutralised to pH 8 using 1 M aqueous sodiumhydroxide and evaporated under reduced pressure to give a white solidwhich was entrained with dichloromethane and methanol (2×95 mL, 1:1,v/v). The solid obtained was removed by suction filtration. The combinedfiltrates were concentrated under reduced pressure to give a gum. Thisimpure gum was entrained with dichloromethane and methanol (10 mL, 9:1,v/v) and the insoluble white solid obtained was removed by suctionfiltration. The filtrate was concentrated under reduced pressure to givesodium(2-{2-[3-(2-aminoethylcarbamoyl)phenoxy]-1-azidoethoxy}ethoxy)acetate(0.76 g, quantitative), as a white foam.

¹H NMR (300 MHz, D₂O): δ 7.31 (m, 3H, 3×ArH), 7.11 (ddd, J=7.8, 2.6, 1.5Hz, 1H, ArH), 4.99 (t, J=4.5 Hz, 1H, CH), 4.15 (m, 2H, ArOCH₂), 3.94 (m,1H, 0.5×OCH₂), 3.79 (m, 3H, 0.5×OCH₂+OCH₂C(O)), 3.58 (m, 4H, OCH₂+CH₂N),3.10 (t, J=5.8 Hz, 2H, CH₂N).

2-(2-{1-Azido-2-[3-({2-[3,5-bis(methoxycarbonyl)benzamido]ethyl}carbamoyl)phenoxy]ethoxy}ethoxy)aceticacid (Fragment 2)

Trimethyl 1,3,5-benzenetricarboxylate (2.00 g, 7.93 mmol) was suspendedin methanol (180 mL) and 1 M aqueous sodium hydroxide solution (7.14 mL)was added. The reaction mixture was stirred at room temperature for 18h. The resulting solution was concentrated under reduced pressure toafford a white solid which was partitioned between dichloromethane (150mL) and saturated aqueous sodium bicarbonate solution (150 mL). Theorganic phase was separated and was extracted with saturated aqueoussodium bicarbonate solution (150 mL) before being discarded. Thecombined aqueous layers were acidified to pH 1-2 using concentratedhydrochloric acid and were extracted with ethyl acetate (2×150 mL). Thecombined organic layers were dried (MgSO₄) and concentrated to give3,5-bis(methoxycarbonyl)benzoic acid (1.66 g, 88%), as a white solid.

3,5-bis(Methoxycarbonyl)benzoic acid (0.80 g, 3.36 mmol) was dissolvedin ethyl acetate (20 mL) and N-hydroxysuccinimide (425 mg, 3.69 mmol)was added followed by N,N′-dicyclohexylcarbodiimide (0.76 g, 3.69 mmol).The reaction mixture was stirred at room temperature for 20 h. Theresulting suspension was filtered over Celite and the filtrate waswashed with saturated aqueous sodium bicarbonate solution (2×100 mL),water (100 mL) and saturated brine (100 mL). The organic layer was dried(MgSO₄) and concentrated under reduced pressure to afford a white glassysolid. This material was purified using a Biotage Isolera automatedchromatography system under normal phase conditions (silica column,gradient of 0→100% ethyl acetate in petrol) with detection at 254 nm togive N-hydroxysuccinimido 3,5-bis(methoxycarbonyl)benzoate (1.06 g,95%), as a white solid.

R_(f) 0.44 (petrol-ethyl acetate, 4:6, v/v).

To a solution of N-hydroxysuccinimido 3,5-bis(methoxycarbonyl)benzoate(0.95 g, 2.83 mmol) and sodium(2-{2-[3-(2-aminoethylcarbamoyl)phenoxy]-1-azidoethoxy}ethoxy)acetate(0.92 g, 2.36 mmol) in anhydrous DMF (15 mL) was added Hunig's base(0.61 g, 0.83 mL, 4.72 mmol) and the reaction mixture was stirred atroom temperature overnight. The resulting mixture was diluted with ethylacetate (20 mL) and quenched with 1 M hydrochloric acid (20 mL). Theaqueous phase was separated, extracted with ethyl acetate (3×30 mL) andthe combined organic phases were washed with water (5×50 mL) andsaturated brine (50 mL). The resulting solution was dried (MgSO₄) beforebeing concentrated under reduced pressure to give a white glassy solid.This material was purified using a Biotage Isolera automatedchromatography system under normal phase conditions (silica column,gradient of 0→50% methanol in dichloromethane) with detection at 254 nmto give2-(2-{1-azido-2-[3-({2-[3,5-bis(methoxycarbonyl)benzamido]ethyl}carbamoyl)phenoxy]ethoxy}ethoxy)aceticacid (Fragment 2) (0.92 g, 66%), as a white glassy solid.

R_(f) 0.37 (dichloromethane-methanol, 83:17, v/v).

¹H NMR (300 MHz, d₆-DMSO) δ 9.42 (br, 1H, NH), 8.99 (br, 1H, NH), 8.66(d, J=1.5 Hz, 2H, 2×ArH), 8.53 (t, J=1.5 Hz, 1H, ArH), 7.50 (m, 1H,ArH), 7.43 (d, J=7.7 Hz, 1H, ArH), 7.33 (t, J=7.9 Hz, 1H, ArH), 7.07(dd, J=8.0, 2.4 Hz, 1H, ArH), 5.21 (t, J=5.0 Hz, 1H, CH), 4.24 (dd,J=10.6, 4.4 Hz, 1H, 0.5×ArOCH₂), 4.07 (dd, J=10.6, 5.9 Hz, 1H,0.5×ArOCH₂), 3.88 (m, 8H, OCH₂+2×OCH₃), 3.84 (m, 1H, 0.5×OCH₂), 3.77 (m,1H, 0.5×OCH₂), 3.62 (m, 2H, OCH₂C(O)), 3.40 (m, 4H, 2×CH₂N).

Pre-Cleavable SCCTP

3′-O-Pivaloyl-5-(3-aminopropynyl)-2′-deoxycytidine trifluoroacetate (379mg, 0.79 mmol) was dissolved in anhydrous DMF (11.1 mL). Hunig's base(292 mg, 391 μL, 2.26 mmol) followed by2-(2-{1-azido-2-[3-({2-[3,5-bis(methoxycarbonyl)benzamido]ethyl}carbamoyl)phenoxy]ethoxy}ethoxy)aceticacid (332 mg, 0.57 mmol) and HBTU (223 mg, 0.59 mmol) were added. Thereaction mixture was stirred at room temperature overnight. Theresulting mixture was diluted with ethyl acetate (50 mL) and washed withwater (50 mL). The aqueous phase was separated and extracted with ethylacetate (2×50 mL). The combined organic phases were washed with water(5×50 mL), saturated brine (50 mL), dried (MgSO₄) and concentrated underreduced pressure to give a pale yellow solid. This solid was purifiedusing a Biotage Isolera automated chromatography system under normalphase conditions (silica column, gradient of 2→16% methanol indichloromethane) with detection at 254 nm to give Pre-Cleavable SCCTP(410 mg, 77%), as a white solid.

R_(f) 0.46 (dichloromethane-methanol, 92:8, v/v).

¹H NMR (300 MHz, DMSO-d₆): δ 9.09 (br t, J=5.8 Hz, 1H, NH), 8.68 (d,J=1.6 Hz, 2H, 2×ArH), 8.63 (br t, J=5.8 Hz, 1H, NH), 8.59 (t, J=1.6 Hz,1H, ArH), 8.24 (t, J=5.4 Hz, 1H, NH), 8.11 (s, 1H, deoxycytidine CH),7.86 (br, 1H, 0.5×NH₂), 7.46 (m, 2H, 2×ArH), 7.38 (t, J=7.8 Hz, 1H,ArH), 7.13 (m, 1H, ArH), 6.94 (br, 1H, 0.5×NH₂), 6.12 (dd, J=8.4, 5.8Hz, 1H, CH), 5.24 (t, J=5.2 Hz, 1H, OH), 5.16 (m, 2H, CHOPiv+CHN), 4.23(dd, J=10.4, 4.5 Hz, 1H, 0.5×ArOCH₂), 4.13 (m, 3H, 0.5×ArOCH₂+OCH₂),3.88 (m, 10H, CH₂N+CH+0.5×OCH₂+2×OCH₃), 3.85 (m, 1H, 0.5×OCH₂), 3.68 (m,2H, OCH₂C(O)), 3.62 (m, 2H, deoxycytidine CH₂), 3.47 (m, 4H, 2×CH₂N),2.17 (m, 2H, deoxycytidine CH₂), 1.16 (s, 9H, 3×CH₃).

Penta-Triethylammonium Cleavable SCCTP

Pre-Cleavable SCCTP (155 mg, 0.17 mmol) was dissolved in 1,4-dioxane(510 μL) and anhydrous pyridine (430 μL) and the flask was evacuated andpurged with a nitrogen atmosphere three times. 1.0 M Salicylchlorophosphite solution in 1,4-dioxane (480 μL, 0.18 mmol) was addedand the reaction mixture was stirred for 10 min. 0.5 M Tributylammoniumpyrophosphate solution in anhydrous DMF (480 μL, 0.25 mmol) andtri-n-butylamine (129 mg, 165 μL, 0.70 mmol) were simultaneously addedand the reaction mixture was stirred for 15 min. 1% Iodine solution inpyridine and water (3.4 mL, 92:8 v/v) was added and the solution wasstirred for 30 min before being quenched with 5% aqueous sodiumthiosulfate solution (100 μL) and concentrated under reduced pressure.The material obtained was purified using a Biotage Isolera automatedchromatography system under reversed-phase conditions (C₁₈ column,gradient of 0→100% acetonitrile in 0.1 M TEAA at pH 8) with detection at292 nm to afford, after freeze-drying, impure protected Cleavable SCCTP(147 mg, 59%) which was used without further purification.

1 M Aqueous sodium hydroxide solution (730 μL) was added to a solutionof protected Cleavable SCCTP (94 mg) in water (800 μL) and stirred atroom temperature for 40 min. The reaction mixture was quenched with 1 Maqueous triethylammonium bicarbonate (TEAB) solution (pH 8.5, 6 mL). Theresulting solution was lyophilized overnight and the material wasdissolved in water (1 mL) to give a concentration of 115 mg/mL. Thissolution was purified by semi-preparative HPLC injecting 60 μL portionsand collecting the eluent containing the pure substance. The combinedfractions were reduced in volume by removing the acetonitrile and mostof the water and finally lyophilized to give penta-triethylammoniumCleavable SCCTP (45 mg, 17% over two steps), as a white solid.

HPLC Conditions:

Column: Phenomenex Luna C18(2), 15 mm×250 mm

Solvent Gradient: 90% 0.1 M aqueous TEAB to 85.6% 0.1 M aqueous TEABover 22 min with the balance being acetonitrile.

Flow Rate: 7.8 mL/min

Temp: 30° C.

Detection: UV at 290 nm

Under these conditions the product had a retention time of ca. 19-21min.

Example 6 Synthesis of Supercharged ATP (SCATP)7-Deaza-7-iodo-2′-deoxyadenosine

6-Chloro-7-iodo-7-deazapurine (0.56 g, 2.00 mmol) was added to a fineslurry of potassium hydroxide (236 mg, 4.20 mmol) andtris(2-(2-methoxyethoxy)ethyl)amine (81 mg, 80 μL, 0.25 mmol) inanhydrous acetonitrile (25 mL) and the resulting mixture was stirred for10 min. 3,5-di-O-(p-toluyl)-2-deoxy-D-ribofuranosyl chloride (0.97 g,2.50 mmol) was added to the reaction mixture and the resulting yellowmixture was stirred for 25 min. The resulting mixture was filteredthrough Celite and the filter cake was washed with acetonitrile (10 mL)followed by dichloromethane (100 mL). The combined filtrates wereconcentrated under reduced pressure and the material obtained waspurified using a Biotage Isolera automated chromatography system undernormal phase conditions (silica column, gradient of 0→40% ethyl acetatein petrol) with detection at 254 nm to give7-deaza-6-chloro-7-iodo-2′-deoxyadenosine (0.94 g, 75%) as a pale yellowsolid.

7-Deaza-6-chloro-7-iodo-2′-deoxyadenosine (0.94 g, 1.49 mmol) wassuspended in a 7 N solution of ammonia in methanol (19 mL) and heatedunder microwave irradiation at 120° C. for 10 h. The resulting solutionwas concentrated under reduced pressure and the residue obtained waspurified using a Biotage Isolera automated chromatography system undernormal phase conditions (silica column, gradient of 0→20% methanol indichloromethane) with detection at 254 nm to give7-deaza-7-iodo-2′-deoxyadenosine (476 mg, 85%), as a white solid.

R_(f) 0.46 (dichloromethane-methanol, 9:1, v/v).

¹H NMR (300 MHz, DMSO-d₆): δ 8.10 (s, 1H, ArH), 7.66 (s, 1H, ArH), 6.68(br s, 2H, NH₂), 6.48 (dd, J=8.3, 5.6 Hz, 1H, CH), 5.26 (d, J=4.6 Hz,1H, OH), 5.05 (t, J=5.4 Hz, 1H, OH), 4.32 (m, 1H, CH), 3.81 (m, 1H, CH),3.53 (m, 2H, OCH₂), 2.44 (m, 1H, 0.5×CH₂), 2.15 (m, 1H, 0.5×CH₂).

5′-O-Dimethoxytrityl-7-deaza-7-iodo-2′-deoxyadenosine

7-Deaza-7-iodo-2′-deoxyadenosine (475 mg, 1.26 mmol) was dissolved inanhydrous pyridine (6 mL) and stirred under nitrogen. After 5 min,4-(dimethylamino)pyridine (8 mg, 0.07 mmol) and triethylamine (18 mg, 23μL, 0.18 mmol) were added to the reaction mixture, followed by4,4′-dimethoxytriphenylmethyl chloride (470 mg, 1.39 mmol). Theresulting reaction mixture was stirred at room temperature overnight andwas quenched with methanol (3 mL) before being concentrated underreduced pressure. The pyridine residues were azeotropically removed withmethanol (3×10 mL) and dichloromethane (10 mL) to give a white solid.This crude material was purified using a Biotage Isolera automatedchromatography system under normal phase conditions (silica column,gradient of 0→10% methanol in dichloromethane) with detection at 254 nmto give 5′-O-dimethoxytrityl-7-deaza-7-iodo-2′-deoxyadenosine (450 mg,53%), as a white solid.

R_(f) 0.36 (dichloromethane-methanol, 9:1, v/v).

¹H NMR (300 MHz, CDCl₃): δ 8.16 (s, 1H, ArH), 7.36 (m, 2H, 2×ArH), 7.25(m, 8H, 8×ArH), 6.78 (m, 4H, 4×ArH), 6.61 (t, J=6.6 Hz, 1H, CH), 5.61(s, 2H, NH₂), 4.54 (m, 1H, CH), 4.01 (m, 1H, CH), 3.73 (s, 6H, 2×OCH₃),3.31 (m, 2H, CH₂), 2.42 (m, 2H, CH₂).

N-3′-Di-pivaloyl-5′-O-dimethoxytrityl-7-deaza-7-iodo-2′-deoxyadenosine

5′-O-Dimethoxytrityl-7-deaza-7-iodo-2′-deoxyadenosine (420 mg, 0.62mmol) was dissolved in anhydrous acetonitrile (9 mL).4-(Dimethylamino)pyridine (15 mg, 0.12 mmol), triethylamine (414 mg, 570μL, 4.09 mmol) and trimethylacetic anhydride (0.76 g, 0.83 mL, 4.09mmol) were added and the reaction mixture was stirred at 50° C. for 48h. The resulting mixture was allowed to cool to room temperature and wasconcentrated under reduced pressure. The impure material wasazeotropically dried with dichloromethane (3×10 mL) to give apale-yellow, glassy solid which was purified using a Biotage Isoleraautomated chromatography system under normal phase conditions (silicacolumn, gradient of 0→10% methanol in dichloromethane) with detection at254 nm to giveN-3′-O-dipivaloyl-5′-O-dimethoxytrityl-7-deaza-7-iodo-2′-deoxyadenosine(0.52 g, quantitative), as a white solid.

R_(f) 0.60 (dichloromethane-methanol, 9:1, v/v).

¹H NMR (300 MHz, CDCl₃): δ 8.92 (s, 1H, NH), 8.74 (s, 1H, ArH), 7.62 (s,1H, ArH), 7.43 (m, 2H, 2×ArH), 7.31 (m, 7H, 7×ArH), 6.86 (m, 4H, 4×ArH),6.78 (m, 1H, CH), 5.54 (m, 1H, CH), 4.19 (m, 1H, CH), 3.81 (s, 6H,2×OCH₃), 3.50 (m, 1H, 0.5×CH₂), 3.41 (m, 1H, 0.5×CH₂), 2.79 (m, 1H,0.5×CH₂), 2.59 (m, 1H, 0.5×CH₂), 1.25 (s, 18H, 6×CH₃).

3′-O-Pivaloyl-5′-O-dimethoxytrityl-7-deaza-7-iodo-2′-deoxyadenosine

N-3′-O-Dipivaloyl-5′-O-dimethoxytrityl-7-deaza-7-iodo-2′-deoxyadenosine(0.52 g, 0.61 mmol) was dissolved in anhydrous methanol (10.5 mL) andHunig's base (119 mg, 160 μL, 0.92 mmol) was added. The reaction mixturewas heated under microwave irradiation at 110° C. for 6 h before beingconcentrated under reduced pressure. The impure material was purifiedusing a Biotage Isolera automated chromatography system under normalphase conditions (silica column, gradient of 0→10% methanol indichloromethane) with detection at 254 nm to give3′-O-pivaloyl-5′-O-dimethoxytrityl-7-deaza-7-iodo-2′-deoxyadenosine (423mg, 91%), as a pale yellow solid.

R_(f) 0.44 (dichloromethane-methanol, 96:4, v/v).

¹H NMR (300 MHz, CDCl₃): δ 8.14 (s, 1H, ArH), 7.30 (m, 10H, 10×ArH),6.76 (m, 4H, 4×ArH), 6.61 (m, 1H, CH), 6.29 (s, 2H, NH₂), 5.43 (m, 1H,CH), 4.09 (m, 1H, CH), 3.73 (s, 6H, 2×OCH₃), 3.37 (m, 2H, CH₂), 2.66 (m,1H, 0.5×CH₂), 2.43 (m, 1H, 0.5×CH₂), 1.17 (s, 9H, 3×CH₃).

3′-O-Pivaloyl-5′-O-dimethoxytrityl-7-deaza-7-(3-Boc-aminoprop-1-yn-1-yl)-2′-deoxyadenosine

3′-O-Pivaloyl-5′-O-dimethoxytrityl-7-deaza-7-iodo-2′-deoxyadenosine (420mg, 0.55 mmol), copper(I) iodide (23 mg, 0.12 mmol) andtetrakis(triphenylphosphine)palladium(0) (64 mg, 0.06 mmol) weredissolved in ethyl acetate (4.2 mL) and the flask was evacuated thenpurged with nitrogen four times. Hunig's base (142 mg, 187 μL, 1.10mmol) and N-Boc-propargylamine (256 mg, 1.65 mmol) were added and theresulting mixture was evacuated and purged with nitrogen before beingleft to stir at room temperature overnight. The reaction mixture wasdiluted with ethyl acetate (25 mL) and washed with 5% aqueous EDTAsolution (10 mL). The layers were separated and the aqueous phase waswashed with ethyl acetate (10 mL). The combined organic layers werewashed with saturated brine (10 mL), dried (MgSO₄) and concentratedunder reduced pressure to give a dark yellow solid. This solid waspurified using a Biotage Isolera automated chromatography system undernormal phase conditions (silica column, gradient of 0→13% methanol indichloromethane) with detection at 254 nm to give3′-O-pivaloyl-5′-O-dimethoxytrityl-7-deaza-7-(3-Boc-aminoprop-1-yn-1-yl)-2′-deoxyadenosine (0.51 g), as a yellow solid.

R_(f) 0.51 (dichloromethane-methanol, 9:1, v/v).

¹H NMR (300 MHz, CDCl₃): δ 8.18 (s, 1H, ArH), 7.26 (m, 10H, 10×ArH),6.76 (m, 4H, 4×ArH), 6.58 (m, 1H, CH), 5.94 (s, 2H, NH₂), 5.37 (m, 1H,CH), 4.94 (m, 1H, CH), 4.10 (m, 1H, NH), 4.01 (d, J=5.7 Hz, 2H, CH₂),3.73 (s, 6H, 2×OCH₃), 3.32 (m, 2H, CH₂), 2.62 (m, 1H, 0.5×CH₂), 2.40 (m,1H, 0.5×CH₂), 1.39 (s, 9H, 3×CH₃), 1.17 (s, 9H, 3×CH₃).

3′-O-Pivaloyl-7-deaza-7-(3-aminoprop-1-yn-1-yl)-2′-deoxyadenosineDi-trifluoroacetate

3′-O-Pivaloyl-5′-O-dimethoxytrityl-7-deaza-7-(3-Boc-aminoprop-1-yn-1-yl)-2′-deoxyadenosine(0.51 g, 0.55 mmol) was dissolved in anhydrous dichloromethane (12 mL)and trifluoroacetic acid (1.70 mL) was added dropwise. The mixture wasthen stirred for 2 h at room temperature and was subsequently dilutedwith dichloromethane (25 mL). The resulting solution was concentratedunder reduced pressure and the residual trifluoroacetic acid wasazeotropically removed with dichloromethane (4×25 mL). The crudematerial obtained was purified using a Biotage Isolera automatedchromatography system under reversed-phase conditions (C₁₈ column,gradient of 0→40% acetonitrile in 0.1% aqueous trifluoroacetic acid)with detection at 254 nm to afford, after freeze-drying, the required3′-O-Pivaloyl-7-deaza-7-(3-aminoprop-1-yn-1-yl)-2′-deoxyadenosinedi-trifluoroacetate (Fragment 1) (257 mg, 76% over two-steps), as anoff-white solid.

¹H NMR (300 MHz, DMSO-d₆): δ 8.29 (m, 7H, ArH+2×NH₃ ⁺), 7.98 (m, 1H,ArH), 6.50 (m, 1H, CH), 5.32 (m, 1H, CH), 4.03 (m, 3H, CH+CH₂), 3.62 (m,2H, CH₂), 2.72 (m, 1H, 0.5×CH₂), 2.40 (m, 1H, 0.5×CH₂), 1.19 (s, 9H,3×CH₃).

3-[(1,3)Dioxolan-2-ylmethoxy]benzoic acid ethyl ester

Ethyl 3-hydroxybenzoate (3.50 g, 21.1 mmol), bromoethyl-1,3-dioxolane(14.1 g, 8.72 mL, 84.3 mmol), potassium carbonate (5.83 g, 42.1 mmol)and sodium iodide (1.26 g, 8.43 mmol) were dissolved in anhydrous DMF(10 mL) and the reaction mixture was stirred at 120° C. for 20 h. Thesuspension was cooled to room temperature and quenched with water (30mL). The aqueous phase was extracted with ethyl acetate (3×50 mL). Theorganic layers were combined, washed with water (5×100 mL) and saturatedbrine (100 mL), before being dried (MgSO₄) and concentrated underreduced pressure to afford an orange oil. This oil was purified using aBiotage Isolera automated chromatography system under normal phaseconditions (silica column, gradient of 0→30% ethyl acetate in petrol)with detection at 254 nm to give 3-[(1,3)dioxolan-2-ylmethoxy]benzoicacid ethyl ester (5.09 g, 96%), as a colorless oil.

R_(f) 0.40 (petrol-ethyl acetate, 75:25, v/v).

¹H NMR (400 MHz, CDCl₃): δ 7.66 (dt, J=7.5, 1.1 Hz, 1H, ArH), 7.59 (dd,J=2.6, 1.5 Hz, 1H, ArH), 7.34 (t, J=7.9 Hz, 1H, ArH), 7.14 (ddd, J=8.3,2.6, 1.0 Hz, 1H, ArH), 5.31 (t, J=4.1 Hz, 1H, CH), 4.36 (q, J=7.2 Hz,2H, OCH₂), 4.02 (m, 6H, OCH₂CH₂O+ArOCH₂), 1.39 (t, J=7.1 Hz, 3H, CH₃).

3-[2-Azido-2-(2-hydroxyethoxy)ethoxy]benzoic acid ethyl ester

To a mixture of 3-[(1,3)dioxolan-2-ylmethoxy]benzoic acid ethyl ester(5.08 g, 20.1 mmol) and azidotrimethylsilane (2.55 g, 2.94 mL, 22.2mmol) was added tin(IV) chloride (336 mg, 151 μL, 1.29 mmol). Thereaction mixture was stirred at room temperature for 2 h. The resultingmixture was diluted with 2% aqueous methanol (60 mL) and stirred for 30min before being concentrated under reduced pressure. The residue wasazeotropically dried with ethanol (2×30 mL) to afford a colorless oil.This oil was purified using a Biotage Isolera automated chromatographysystem under normal phase conditions (silica column, gradient of 0→70%ethyl acetate in petrol) with detection at 254 nm to give3-[2-azido-2-(2-hydroxyethoxy)ethoxy]benzoic acid ethyl ester (3.59 g,59%), as a colorless oil.

R_(f) 0.41 (petrol-ethyl acetate, 53:47, v/v).

¹H NMR (300 MHz, CDCl₃): δ 7.69 (dt, J=7.7, 1.2 Hz, 1H, ArH), 7.59 (dd,J=2.6, 1.5 Hz, 1H, ArH), 7.36 (t, J=8.0 Hz, 1H, ArH), 7.13 (ddd, J=8.3,2.7, 1.0 Hz, 1H, ArH), 4.89 (t, J=5.1 Hz, 1H, CH), 4.38 (q, J=7.1 Hz,2H, OCH₂), 4.19 (m, 2H, ArOCH₂), 4.00 (m, 1H, 0.5×OCH₂), 3.80 (m, 3H,0.5×OCH₂+OCH₂), 1.40 (t, J=7.1 Hz, 3H, CH₃).

3-[2-Azido-2-(2-hydroxyethoxy)ethoxy]benzoic acid

3-[2-Azido-2-(2-hydroxyethoxy)ethoxy]benzoic acid ethyl ester (3.51 g,11.9 mmol) was dissolved in ethanol (30 mL) and 4 M aqueous sodiumhydroxide (30 mL) was added. The mixture was stirred at room temperaturefor 3.5 h and the volume was then reduced by ¾ under vacuum. Theresulting mixture was diluted with water (50 mL) and acidified to pH 1-2with 2 M hydrochloric acid. This mixture was extracted withdichloromethane (3×100 mL). The combined organic phases were washed withsaturated brine (150 mL), dried (MgSO₄) and concentrated under reducedpressure to afford 3-[2-azido-2-(2-hydroxyethoxy)ethoxy]benzoic acid(3.39 g, quantitative), as a colorless oil which was used withoutfurther purification.

¹H NMR (300 MHz, CDCl₃): δ 7.76 (dt, J=7.7, 1.2 Hz, 1H, ArH), 7.65 (dd,J=2.4, 1.4 Hz, 1H, ArH), 7.41 (t, J=8.0 Hz, 1H, ArH), 7.19 (ddd, J=8.2,2.7, 1.0 Hz, 1H, ArH), 4.90 (t, J=5.1 Hz, 1H, CH), 4.21 (m, 2H, ArOCH₂),4.03 (m, 1H, 0.5×OCH₂), 3.81 (m, 3H, 0.5×OCH₂+OCH₂).

3-[2-Azido-2-(2-ethoxycarbonylmethoxyethoxy)ethoxy]benzoic acid

To an ice-cold solution of 3-[2-azido-2-(2-hydroxyethoxy)ethoxy]benzoicacid (1.42 g, 5.31 mmol) in anhydrous THF (18 mL) was added sodiumhydride (0.64 g, 15.9 mmol) and the mixture was stirred at 0° C. for 10min. Ethyl bromoacetate (1.95 g, 1.30 mL, 11.7 mmol) was added and thereaction mixture was allowed to warm up to room temperature over 5 h.The resulting mixture was quenched with ice-water (20 mL) and washedwith dichloromethane (2×70 mL). The aqueous layer was acidified to pH1-2 using 2 M hydrochloric acid and was extracted with dichloromethane(3×70 mL). The combined organic layers were dried (MgSO₄) andconcentrated under reduced pressure to afford a colorless oil. Thismaterial was purified using a Biotage Isolera automated chromatographysystem under normal phase conditions (silica column, gradient of 0→60%ethyl acetate in dichloromethane) with detection at 254 nm to give3-[2-azido-2-(2-ethoxycarbonylmethoxyethoxy)ethoxy]benzoic acid (0.62 g,33%), as a colorless oil.

R_(f) 0.45 (ethyl acetate-dichloromethane, 4:6 v/v).

¹H NMR (300 MHz, CDCl₃): δ 7.75 (dt, J=7.7, 1.2 Hz, 1H, ArH), 7.64 (dd,J=2.7, 1.5 Hz, 1H, ArH), 7.40 (t, J=8.0 Hz, 1H, ArH), 7.19 (ddd, J=8.3,2.7, 1.0 Hz, 1H, ArH), 4.95 (dd, J=5.5, 4.6 Hz, 1H, CH), 4.14 (m, 7H,ArOCH₂+0.5×OCH₂+OCH₂+OCH₂C(O)), 3.86 (m, 3H, 0.5×OCH₂+OCH₂), 1.28 (t,J=7.2 Hz, 3H, CH₃).

N-(2-Aminoethyl)-2,2,2-trifluoroacetamide Trifluoroacetate

To an ice-cold solution of N-Boc-ethylenediamine (2.08 g, 2.05 mL, 13.0mmol) in anhydrous THF (8 mL) was slowly added ethyl trifluoroacetate(1.85 g, 1.55 mL, 13.0 mmol) and the reaction mixture was stirred atroom temperature overnight. The resulting solution was concentratedunder reduced pressure to give tert-butyl[2-(2,2,2-trifluoroacetamido)ethyl]carbamate (3.30 g, 99%), as a whitesolid which was used without further purification.

tert-Butyl [2-(2,2,2-trifluoroacetamido)ethyl]carbamate (1.04 g, 4.06mmol) was dissolved in trifluoroacetic acid (5 mL) and stirred at roomtemperature for 30 min. The resulting mixture was concentrated underreduced pressure and the residual trifluoroacetic acid wasazeotropically removed with chloroform (3×10 mL). The material obtainedwas dried in vacuo at 50° C. for 2 h to giveN-(2-aminoethyl)-2,2,2-trifluoroacetamide trifluoroacetate (1.07 g,99%), as a yellow oil which was used without further purification.

¹H NMR (300 MHz, DMSO-d₆): δ 9.58 (br t, 1H, NH), 7.99 (br s, 3H, NH₃⁺), 3.43 (m, 2H, CH₂N), 2.97 (m, 2H, CH₂N).

[2-(1-Azido-2-{3-[2-(2,2,2-trifluoroacetamido)ethylcarbamoyl]phenoxy}ethoxy)ethoxy]aceticacid ethyl ester

To a solution of N-(2-aminoethyl)-2,2,2-trifluoroacetamidetrifluoroacetate (1.31 g, 4.86 mmol) in anhydrous DMF (25 mL) was addedHunig's base (1.57 g, 2.12 mL, 12.1 mmol),3-[2-azido-2-(2-ethoxycarbonylmethoxyethoxy)ethoxy]benzoic acid (1.43 g,4.05 mmol) and PyBOP (2.32 g, 4.45 mmol). The reaction mixture wasstirred at room temperature for 20 h before being diluted with ethylacetate (20 mL) and quenched with 1 M hydrochloric acid (40 mL). Theaqueous phase was extracted with ethyl acetate (3×50 mL). The combinedorganic layers were washed with water (5×100 mL), saturated brine (100mL), dried (MgSO₄) and concentrated under reduced pressure to give ayellow oil. This oil was purified using a Biotage Isolera automatedchromatography system under normal phase conditions (silica column,gradient of 0→90% ethyl acetate in dichloromethane) with detection at254 nm to give[2-(1-azido-2-{3-[2-(2,2,2-trifluoroacetamido)ethylcarbamoyl]phenoxy}ethoxy)ethoxy]aceticacid ethyl ester (1.75 g, 88%) as a colorless oil.

R_(f) 0.48 (dichloromethane-ethyl acetate, 1:1, v/v).

¹H NMR (300 MHz, CDCl₃): δ 8.01 (br, 1H, NH), 7.39 (m, 3H, 3×ArH), 7.10(dt, J=5.8, 3.0 Hz, 1H, ArH), 6.97 (br, 1H, NH), 4.93 (t, J=5.1 Hz, 1H,CH), 4.18 (m, 6H, ArOCH₂+OCH₂+OCH₂C(O)), 4.04 (m, 1H, 0.5×OCH₂), 3.87(m, 1H, 0.5×OCH₂), 3.81 (m, 2H, OCH₂), 3.69 (m, 2H, CH₂N), 3.62 (m, 2H,CH₂N), 1.30 (t, J=7.1 Hz, 3H, CH₃).

Sodium(2-{2-[3-(2-aminoethylcarbamoyl)phenoxy]-1-azidoethoxy}ethoxy)acetate

[2-(1-Azido-2-{3-[2-(2,2,2-trifluoroacetamido)ethylcarbamoyl]phenoxy}ethoxy)ethoxy]aceticacid ethyl ester (0.90 g, 1.83 mmol) was dissolved in ethanol (7 mL) and4 M aqueous sodium hydroxide (7 mL) was added. The resulting mixture wasstirred at room temperature for 2.5 h before being concentrated underreduced pressure. The material obtained was dissolved in water (55 mL)and washed with dichloromethane (2×48 mL). The aqueous layer wasacidified to pH 2 using 1 M hydrochloric acid and washed withdichloromethane (2×50 mL). The organic layer was removed and discardedwhile the aqueous layer was neutralised to pH 8 using 1 M aqueous sodiumhydroxide and evaporated under reduced pressure to give a white solidwhich was entrained with dichloromethane and methanol (2×95 mL, 1:1,v/v). The solid obtained was removed by suction filtration. The combinedfiltrates were concentrated under reduced pressure to give a gum. Thiscrude gum was entrained with dichloromethane and methanol (10 mL, 9:1,v/v) and the insoluble white solid obtained was removed by suctionfiltration. The filtrate was concentrated under reduced pressure to givesodium (2-{2-[3-(2-aminoethylcarbamoyl)phenoxy]-1-azidoethoxy}ethoxy)acetate (0.76 g,quantitative), as a white foam.

¹H NMR (300 MHz, D₂O): δ 7.31 (m, 3H, 3×ArH), 7.11 (ddd, J=7.8, 2.6, 1.5Hz, 1H, ArH), 4.99 (t, J=4.5 Hz, 1H, CH), 4.15 (m, 2H, ArOCH₂), 3.94 (m,1H, 0.5×OCH₂), 3.79 (m, 3H, 0.5×OCH₂+OCH₂C(O)), 3.58 (m, 4H, OCH₂+CH₂N),3.10 (t, J=5.8 Hz, 2H, CH₂N).

N-Hydrosuccinimido3,5-bis{[(S)-1,5-dimethoxy-1,5-dioxopentan-2-yl]carbamoyl}benzoate

Trimethyl 1,3,5-benzenetricarboxylate (4.00 g, 15.9 mmol) was suspendedin methanol (360 mL) and 1 M aqueous sodium hysroxide (14.3 mL, 14.3mmol) was added. The mixture was vigorously stirred at room temperaturefor 18 h. The resulting solution was concentrated under reduced pressureto afford a white solid which was partitioned between dichloromethane(300 mL) and saturated aqueous sodium bicarbonate solution (300 mL). Theorganic phase was separated and was extracted with saturated aqueoussodium bicarbonate solution (300 mL) before being discarded. Thecombined aqueous extracts were acidified to pH 1-2 using concentratedhydrochloric acid and were extracted with ethyl acetate (2×300 mL). Thecombined organic layers were dried (MgSO₄) and concentrated to give3,5-bis(methoxycarbonyl)benzoic acid (3.10 g, 82%), as a white solid.

To an ice-cold suspension of 3,5-bis(methoxycarbonyl)benzoic acid (1.35g, 5.67 mmol) in anhydrous pyridine (15 mL) was added tosyl chloride(1.84 g, 9.63 mmol) and tert-butanol (0.50 g, 0.64 mL, 6.80 mmol) andthe reaction mixture was stirred at room temperature for 20 h. Theresulting suspension was diluted with ethyl acetate (75 mL) and a 20%aqueous solution of citric acid (75 mL). The aqueous layer was extractedwith diethyl ether (3×100 mL) and the combined organic layers werewashed with a 20% aqueous solution of citric acid (100 mL), 1 Mhydrochloric acid (100 mL), water (3×100 mL) and saturated brine (100mL), before being dried (MgSO₄) and concentrated to give a pale yellowsolid. This material was purified using a Biotage Isolera automatedchromatography system under normal phase conditions (silica column,gradient of 0→50% ethyl acetate in petrol) with detection at 254 nm togive 1-(tert-butyl) 3,5-dimethyl benzene-1,3,5-tricarboxylate (1.10 g,66%), as a white solid.

R_(f) 0.32 (ethyl acetate-petrol, 3:7, v/v).

1-(tert-Butyl) 3,5-dimethyl benzene-1,3,5-tricarboxylate (0.50 g, 1.70mmol) was suspended in anhydrous THF (25 mL) and 0.1 M aqueous sodiumhydroxide (15 mL) was added. The resulting suspension was stirred atroom temperature for 2 h. The pH of the reaction mixture was adjusted to11 by addition of 1 M aqueous sodium hydroxide and the resulting mixturewas stirred for 1 h. The reaction mixture was neutralised with 1 Mhydrochloric acid before being concentrated to ⅓ of the volume. Thisresulting mixture was diluted with water (15 mL) and the pH was adjustedto 2 with addition of 2 M hydrochloric acid. The precipitate formed wascollected by suction filtration and azeotropically dried withacetonitrile (3×10 mL) to give an off-white solid. This reactionsequence was repeated due to the presence of mono-methyl ester left inthe resulting solid to afford 5-(tert-butoxycarbonyl)isophthalic acid(428 mg, 95%), as a white solid.

To a solution of H-Glu(OMe)-OMe hydrochloride (1.27 g, 5.99 mmol) andHunig's base (1.03 g, 1.39 mL, 7.98 mmol) in anhydrous DMF (7 mL) wasadded a solution of 5-(tert-butoxycarbonyl)isophthalic acid (0.53 g,2.00 mmol) in anhydrous DMF (8 mL), followed by PyBOP (2.29 g, 4.39mmol) and the reaction mixture was stirred at room temperature for 20 h.The resulting mixture was diluted with ethyl acetate (40 mL) andquenched with 1 M aqueous sodium hydroxide (50 mL). The aqueous layerwas separated and was then extracted with ethyl acetate (3×25 mL). Thecombined organic layers were washed with water (70 mL), saturatedaqueous sodium bicarbonate solution (70 mL), water (5×70 mL) andsaturated brine (70 mL) before being dried (MgSO₄) and concentratedunder reduced pressure to give a white solid. This solid was purifiedusing a Biotage Isolera automated chromatography system under normalphase conditions (silica column, gradient of 0→70% ethyl acetate indichloromethane) with detection at 254 nm to give tetramethyl2,2′-{[5-(tert-butoxycarbonyl)isophthaloyl]bis(azanediyl)}(2S,2′S)-diglutarate(0.69 g, 60%), as a white solid.

R_(f) 0.44 (dichloromethane-ethyl acetate, 6:4, v/v).

Tetramethyl2,2′-{[5-(tert-butoxycarbonyl)isophthaloyl]bis(azanediyl)}(2S,2′S)-diglutarate(0.67 g, 1.15 mmol) was stirred in trifluoroacetic acid (9 mL) for 1 h.The solution was concentrated under reduced pressure and the residualtrifluoroacetic acid was azeotropically removed with chloroform (5×10mL) and diethyl ether (3×10 mL) to give5-bis{[(S)-1,5-dimethoxy-1,5-dioxopentan-2-yl]carbamoyl}benzoic acid(0.61 g, 99%), as a white foam.

This reaction was repeated to yield further batches of material.

5-bis{[(S)-1,5-Dimethoxy-1,5-dioxopentan-2-yl]carbamoyl}benzoic acid(0.64 g, 1.23 mmol) was dissolved in ethyl acetate (20 mL) andN-hydroxysuccinimide (156 mg, 1.35 mmol) was added followed byN,N′-dicyclohexylcarbodiimide (279 mg, 1.35 mmol). The resulting mixturewas stirred at room temperature for 20 h. The suspension was filteredthrough Celite and the filtrate was washed with saturated aqueous sodiumbicarbonate solution (2×30 mL), water (2×30 mL) and saturated brine (30mL). The organic layer was dried (MgSO₄) and concentrated under reducedpressure to afford a white foam. This material was purified using aBiotage Isolera automated chromatography system under normal phaseconditions (silica column, gradient of 0→100% ethyl acetate in petrol)with detection at 254 nm to give N-hydrosuccinimido3,5-bis{[(S)-1,5-dimethoxy-1,5-dioxopentan-2-yl]carbamoyl}benzoate (0.63g, 79%), as a white solid.

R_(f) 0.44 (petrol-ethyl acetate, 4:6, v/v).

¹H NMR (300 MHz, DMSO-d₆): δ 9.32 (d, J=7.5 Hz, 2H, 2×NH), 8.77 (m, 1H,ArH), 8.71 (m, 2H, 2×ArH), 4.53 (m, 2H, 2× α-CH), 3.67 (s, 6H, 2×OCH₃),3.59 (s, 6H, 2×OCH₃), 2.94 (s, 4H, 2×CH₂-su), 2.46 (m, 4H, 2×CH₂), 2.07(m, 4H, 2×CH₂).

2-{2-[1-azido-2-(3-{[2-(3,5-bis{[(S)-1,5-dimethoxy-1,5-dioxopentan-2-yl]carbamoyl}benzamido)ethyl]carbamoyl}phenoxy)ethoxy]ethoxy}aceticacid

To a solution of N-hydroxysuccinimido 3,5-bis(methoxycarbonyl)benzoate(0.78 g, 1.25 mmol) and sodium(2-{2-[3-(2-aminoethylcarbamoyl)phenoxy]-1-azidoethoxy}ethoxy)acetate(445 mg, 1.14 mmol) in anhydrous DMF (7 mL) was added Hunig's base (295mg, 400 μL, 2.28 mmol) and the reaction mixture was stirred at roomtemperature for 20 h. The resulting mixture was diluted with ethylacetate (10 mL) and quenched with 1 M hydrochloric acid (20 mL). Theaqueous phase was separated and extracted with ethyl acetate (3×30 mL)and the combined organic phases were washed with water (5×50 mL) andsaturated brine (50 mL). The resulting solution was dried (MgSO₄) beforebeing concentrated under reduced pressure to give a white foam. Thiscrude material was purified using a Biotage Isolera automatedchromatography system under normal phase conditions (silica column,gradient of 0→50% methanol in dichloromethane) with detection at 254 nmto give2-{2-[1-azido-2-(3-{[2-(3,5-bis{[(S)-1,5-dimethoxy-1,5-dioxopentan-2-yl]carbamoyl}benzamido)ethyl]carbamoyl}phenoxy)ethoxy]ethoxy}aceticacid (Fragment 2) (0.83 g, 83%), as a white solid.

R_(f) 0.43 (dichloromethane-methanol, 83:17, v/v).

¹H NMR (300 MHz, DMSO-d₆): δ 9.26 (br s, 1H, NH), 9.17 (d, J=7.3 Hz, 2H,2×NH), 8.95 (br s, 1H, NH), 8.55 (m, 2H, 2×ArH), 8.47 (m, 1H, ArH), 7.55(m, 1H, ArH), 7.49 (m, 1H, ArH), 7.37 (t, J=7.9 Hz, 1H, ArH), 7.11 (dd,J=7.9, 1.5 Hz, 1H, ArH), 5.20 (t, J=5.0 Hz, 1H, CH), 4.49 (m, 2H, 2×α-CH), 4.25 (dd, J=10.7, 4.5 Hz, 1H, 0.5×CH₂), 4.14 (dd, J=10.7, 5.3 Hz,1H, 0.5×CH₂), 3.89 (m, 2H, CH₂), 3.80 (m, 2H, CH₂), 3.65 (s, 8H,2×OCH₃+CH₂), 3.59 (s, 6H, 2×OCH₃), 3.49 (br, 4H, 2×CH₂), 2.47 (m, 4H,2×CH₂), 2.07 (m, 4H, 2×CH₂).

Pre-Cleavable SCATP

3′-O-Pivaloyl-5-(3-aminopropynyl)-2′-deoxyuridine di-trifluoroacetate(366 mg, 0.60 mmol) was dissolved in anhydrous DMF (12 mL). Hunig's base(295 mg, 390 μL, 2.28 mmol) followed by2-(2-{1-azido-2-[3-({2-[3,5-bis(methoxycarbonyl)benzamido]ethyl}carbamoyl)phenoxy]ethoxy}ethoxy)aceticacid (399 mg, 0.46 mmol) and HBTU (182 mg, 0.48 mmol) were added. Thereaction mixture was stirred at room temperature for 20 h. The resultingmixture was diluted with ethyl acetate (20 mL) and washed with water (20mL). The aqueous phase was separated and extracted with ethyl acetate(2×20 mL). The combined organic phases were washed with water (5×40 mL)and saturated brine (40 mL), before being dried (MgSO₄) and concentratedunder reduced pressure to give an orange solid. This solid was purifiedusing a Biotage Isolera automated chromatography system under normalphase conditions (silica column, gradient of 0→30% methanol in ethylacetate) with detection at 254 nm to afford Pre-Cleavable SCATP (307 mg,54%), as an off-white solid.

R_(f) 0.41 (ethyl acetate-methanol, 9:1, v/v).

¹H NMR (300 MHz, DMSO-d₆): δ 9.10 (d, J=7.3 Hz, 2H, 2×NH), 8.92 (br s,1H, NH), 8.65 (br s, 1H, NH), 8.48 (s, 2H, 2×ArH), 8.37 (t, J=5.4 Hz,1H, ArH), 8.10 (s, 1H, ArH), 7.75 (s, 1H, ArH), 7.46 (m, 2H, 2×ArH),7.36 (t, J=7.8 Hz, 1H, ArH), 7.13 (dd, J=7.7, 2.1 Hz, 1H, ArH), 6.45(dd, J=9.0, 5.5 Hz, 1H, CH), 5.31 (m, 2H, OH+CH), 5.15 (t, J=4.5 Hz, 1H,CHN₃), 4.51 (m, 2H, 2× α-CH), 4.22 (dd, J=10.5, 4.6 Hz, 1H, 0.5×CH₂),4.14 (m, 3H, 0.5×CH₂+CH₂), 3.97 (m, 5H, CH+2×CH₂), 3.86 (m, 2H, CH₂),3.67 (br, 8H, 2×OCH₃+CH₂), 3.59 (s, 6H, 2×OCH₃), 3.46 (br, 4H, 2×NCH₂),2.72 (m, 1H, 0.5×CH₂), 2.47 (m, 4H, 2×CH₂), 2.35 (m, 1H, 0.5×CH₂), 2.07(m, 4H, 2×CH₂), 1.19 (s, 9H, 3×CH₃).

Hepta-Triethylammonium Cleavable SCATP

Pre-Cleavable SCATP (300 mg, 0.24 mmol) was dissolved in 1,4-dioxane(0.73 mL) and anhydrous pyridine (245 μL) and the flask was evacuatedand purged with a nitrogen atmosphere five times. 1.0 M Salicylchlorophosphite solution in 1,4-dioxane (270 μL, 0.27 mmol) was addedand the reaction mixture was stirred for 10 min. 0.5 M Tributylammoniumpyrophosphate solution in anhydrous DMF (0.73 mL, 0.36 mmol) andtributylamine (187 mg, 240 μL, 1.01 mmol) were added and the reactionmixture was stirred for 20 min. 1% Iodine solution in pyridine and water(4.9 mL, 98:2 v/v) were added and the solution was stirred for 15 minbefore being quenched with 5% aqueous sodium thiosulfate solution (0.60mL) and concentrated under reduced pressure. The material obtained waspurified using a Biotage Isolera automated chromatography system underreversed-phase conditions (C₁₈ column, gradient of 0→40% acetonitrile in0.1 M TEAA at pH 8) with detection at 279 nm to afford, afterfreeze-drying, impure protected Cleavable SCATP (178 mg, 42%) which wasused without further purification.

Protected Cleavable SCATP (175 mg) was dissolved in water (1.23 mL) andthe resulting solution was stirred for 15 min before addition of 1 Maqueous sodium hydroxide (1.47 mL). The mixture was stirred at roomtemperature for 40 min and was diluted with 1 M aqueous triethylammoniumbicarbonate (TEAB) solution (pH 8, 3 mL). The resulting solution waslyophilized overnight (250 mg) and the material was dissolved in water(3.6 mL) to give a concentration of 69.4 mg/mL. This solution waspurified by semi-preparative HPLC injecting 100 μL portions andcollecting the eluent containing the pure substance. The combinedfractions were reduced in volume by removing the acetonitrile and mostof the water and finally lyophilized to give hepta-triethylammoniumCleavable SCATP (150 mg, 75%), as a white solid.

HPLC Conditions:

Column: Phenomenex Luna C18(2), 15 mm×250 mm

Solvent Gradient: 90% 0.1 M aqueous TEAB (pH 7.0) to 85.5% 0.1 M aqueousTEAB (pH 7.0) over 30 min with the balance being acetonitrile.

Flow Rate: 7.8 mL/min

Temp: 30° C.

Detection: UV at 277 nm

Under these conditions the product had a retention time of ca. 23-30min.

Example 7 Synthesis of Supercharged GTP (SCGTP)6-O-Methyl-7-deaza-2′-deoxyguanosine

A fine slurry of powdered potassium hydroxide (1.14 g, 20.4 mmol) andtris-(2-(2-methoxyethoxy)ethyl)amine (TDA-1) (96 mg, 95 μL, 0.30 mmol)was stirred in anhydrous acetonitrile (40 mL) for 15 min at roomtemperature. 2-Amino-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (1.00 g, 5.93mmol) was added and the reaction mixture was stirred for 10 min.3,5-Di-O-(p-tolyl)-2-deoxy-D-ribofuranosyl chloride (2.39 g, 5.99 mmol)was added and the reaction mixture was stirred for 30 min. The resultingmixture was filtered through Celite, the filter cake was washed withacetonitrile (10 mL) and the filtrate concentrated to give a brownsolid. This material was purified using a Biotage Isolera automatedchromatography system under normal phase conditions (silica column,gradient of 0→11% ethyl acetate in dichloromethane) with detection at254 nm to give4-chloro-7-((4S,5R)-4-(p-tolyloxy)-5-((p-tolyloxy)methyl)tetrahydrofuran-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-2-amine(1.98 g, 64%), as an off-white foam.

R_(f) 0.37 (dichloromethane-ethyl acetate, 96:4, v/v).

To a suspension of4-chloro-7-((4S,5R)-4-(p-tolyloxy)-5-((p-tolyloxy)methyl)tetrahydrofuran-2-yl)-7H-pyrrolo[2,3-d]pyrimidin-2-amine(1.98 g, 3.80 mmol) in anhydrous methanol (35 mL) was added a 0.5 Msodium methoxide solution in methanol (84 mL, 41.8 mmol) and the mixturewas heated at 65° C. for 2 h. The resulting solution was cooled to roomtemperature and concentrated under reduced pressure to give an off-whitesolid. This residual material was purified using a Biotage Isoleraautomated chromatography system under normal phase conditions (silicacolumn, gradient of 0→25% methanol in dichloromethane) with detection at254 nm to give 6-O-methyl-7-deaza-2′-deoxyguanosine (0.89 g, 84%), as awhite foam.

R_(f) 0.50 (dichloromethane-methanol, 90:10, v/v).

¹H NMR (300 MHz, DMSO-d₆) δ 7.10 (d, J=3.7 Hz, 1H, CH), 6.40 (m, 1H,CHN), 6.27 (d, J=3.7 Hz, 1H, CH), 6.22 (br s, 2H, NH₂), 5.22 (d, J=3.8Hz, 1H, OH), 4.94 (t, J=5.6 Hz, 1H, OH), 4.28 (m, 1H, CH), 3.91 (s, 3H,CH₃), 3.77 (m, 1H, CH), 3.50 (m, 2H, CH₂), 2.39 (m, 1H, 0.5×CH₂), 2.09(m, 1H, 0.5×CH₂).

6-O-Methyl-2,3′,5′-tripivaloyl-7-deaza-2′-deoxyguanosine

To an ice-cold solution of 6-O-methyl-7-deaza-2′-deoxyguanosine (0.89 g,3.18 mmol) in anhydrous pyridine (33 mL) was added isobutyryl chloride(3.05 g, 3.00 mL, 28.6 mmol) dropwise and the mixture was stirred for 1h at this temperature. Methanol (2 mL) was added and the mixture wasstirred for 15 min at 0° C. The suspension was concentrated and theresidue was partitioned between saturated aqueous sodium bicarbonatesolution (50 mL) and ethyl acetate (50 mL). The layers were separatedand the aqueous layer was extracted with ethyl acetate (2×50 mL). Thecombined organic layers were washed with saturated aqueous sodiumbicarbonate solution (50 mL), saturated brine (50 mL), dried (MgSO₄) andconcentrated to give a yellow oil. This crude material was purifiedusing a Biotage Isolera automated chromatography system under normalphase conditions (silica column, gradient of 0→58% ethyl acetate inpetrol) with detection at 254 nm to give6-O-methyl-2,3′,5′-tripivaloyl-7-deaza-2′-deoxyguanosine (1.46 g, 94%),as a colorless oil.

R_(f) 0.47 (petrol-ethyl acetate, 34:66, v/v).

¹H NMR (300 MHz, Methanol-d₄) δ 7.31 (d, J=3.7 Hz, 1H, CH), 6.68 (dd,J=8.7, 5.8 Hz, 1H, CHN), 6.54 (d, J=3.7 Hz, 1H, CH), 5.43 (m, 1H, CH),4.38 (m, 2H, CH₂), 4.26 (m, 1H, CH), 4.01 (s, 3H, CH₃), 2.90 (m, 2H,2×CH), 2.65 (m, 2H, CH+0.5×CH₂), 2.53 (m, 1H, 0.5×CH₂), 1.24 (m, 12H,4×CH₃), 1.17 (m, 6H, 2×CH₃).

6-O-Methyl-2,3′,5′-tripivaloyl-7-deaza-7-iodo-2′-deoxyguanosine

To a solution of6-O-methyl-2,3′,5′-tripivaloyl-7-deaza-2′-deoxyguanosine (1.13 g, 2.31mmol) in anhydrous DMF (18 mL) was added N-iodosuccinimide (0.55 g, 2.42mmol) and the mixture was stirred for 3 h at room temperature. Theresulting red solution was concentrated to give a residual red oil thatwas partitioned between diethyl ether (20 mL) and saturated aqueoussodium bicarbonate solution (20 mL). The layers were separated and theaqueous layer was extracted with diethyl ether (2×25 mL). The combinedorganic layers were washed with water (4×50 mL), saturated brine (50mL), dried (MgSO₄) and concentrated to give a brown solid. This materialwas purified using a Biotage Isolera automated chromatography systemunder normal phase conditions (silica column, gradient of 7→49% ethylacetate in petrol) with detection at 254 nm to give6-O-methyl-2,3′,5′-tripivaloyl-7-deaza-7-iodo-2′-deoxyguanosine (1.08 g,76%), as an off-white solid.

R_(f) 0.37 (petrol-ethyl acetate, 72:28, v/v).

¹H NMR (300 MHz, Methanol-d₄) δ 7.46 (s, 1H, CH), 6.65 (m, 1H, CHN),5.43 (m, 1H, CH), 4.37 (m, 2H, CH₂), 4.26 (m, 1H, CH), 4.11 (s, 3H,CH₃), 2.85 (m, 2H, CH+0.5×CH₂), 2.62 (m, 3H, 2×CH+0.5×CH₂), 1.21 (m,18H, 6×CH₃).

7-Deaza-7-iodo-2′-deoxyguanosine

A suspension of6-O-methyl-2,3′,5′-tripivaloyl-7-deaza-7-iodo-2′-deoxyguanosine (1.40 g,2.27 mmol) in 2 M aqueous sodium hydroxide (65 mL) and 1,4-dioxane (9mL) was heated at 95° C. overnight. The mixture was allowed to cool toroom temperature and the 1,4-dioxane was removed under vacuum. Theaqueous solution was neutralised with 2 M hydrochloric acid to pH=7 andconcentrated to dryness to give a white solid. This residual solid wassuspended in water (100 mL), the solid was collected by suctionfiltration, washed with acetonitrile (20 mL) and dried in vacuo at 40°C. for 2 h to give 7-deaza-7-iodo-2′-deoxyguanosine (0.90 g), as a whitesolid that was used without further purification.

¹H NMR (300 MHz, DMSO-d₆) δ 10.52 (s, 1H, NH), 7.12 (s, 1H, CH), 6.35(br s, 2H, NH₂), 6.26 (dd, J=8.5, 5.7 Hz, 1H, CHN), 5.21 (d, J=3.7 Hz,1H, OH), 4.92 (t, J=5.4 Hz, 1H, OH), 4.27 (m, 1H, CH), 3.75 (m, 1H, CH),3.49 (m, 2H, CH₂), 2.32 (m, 1H, 0.5×CH₂), 2.05 (m, 1H, 0.5×CH₂).

5′-O-(tert-Butyldimethylsilyl)-7-deaza-7-iodo-2′-deoxyguanosine

To a solution of 7-deaza-7-iodo-2′-deoxyguanosine (0.90 g, 2.29 mmol) inanhydrous DMF (12 mL) was added imidazole (375 mg, 5.51 mmol) followedby tert-butyldimethylsilyl chloride (398 mg, 2.64 mmol) and the mixturewas stirred for 20 h at room temperature. The resulting yellow solutionwas concentrated under reduced pressure to give an orange oil. Thiscrude material was purified using a Biotage Isolera automatedchromatography system under normal phase conditions (silica column,gradient of 7→36% methanol in dichloromethane) with detection at 254 nmto give 5′-O-(tert-butyldimethylsilyl)-7-deaza-7-iodo-2′-deoxyguanosine(0.81 g, 70% over two steps), as an off-white solid.

R_(f) 0.43 (dichloromethane-methanol, 88:12, v/v).

¹H NMR (300 MHz, DMSO-d₆) δ 10.48 (s, 1H, NH), 7.05 (s, 1H, CH), 6.35(br s, 2H, NH₂), 6.27 (dd, J=8.2, 5.8 Hz, 1H, CHN), 5.25 (d, J=3.7 Hz,1H, OH), 4.26 (m, 1H, CH), 3.79 (m, 1H, CH), 3.70 (m, 2H, CH₂), 2.25 (m,1H, 0.5×CH₂), 2.10 (m, 1H, 0.5×CH₂), 0.89 (s, 9H, TBDMS tert-butyl),0.08 (s, 3H, TBDMS CH₃), 0.07 (s, 3H, TBDMS CH₃).

3′-O-Pivaloyl-5′-O-(tert-butyldimethylsilyl)-7-deaza-7-iodo-2′-deoxyguanosine

To a solution of5′-O-(tert-butyldimethylsilyl)-7-deaza-7-iodo-2′-deoxyguanosine (0.80 g,1.58 mmol) in anhydrous pyridine (20 mL) was added4-dimethylamino-pyridine (DMAP) (39 mg, 0.32 mmol) followed bytrimethylacetic anhydride (442 mg, 481 μL, 2.37 mmol) and the mixturewas heated at 85° C. overnight. A further equivalent of trimethylaceticanhydride (295 mg, 321 μL, 1.58 mmol) and dimethylaminopyridine (DMAP)(39 mg, 0.32 mmol) was added and the reaction mixture was heated at 85°C. for 2 days. The resulting solution was cooled to room temperature andconcentrated to dryness. The residue was partitioned between ethylacetate (25 mL) and saturated aqueous sodium bicarbonate solution (25mL). The layers were separated and the aqueous layer was extracted withethyl acetate (2×20 mL). The combined organic layers were washed withsaturated brine (40 mL), dried (MgSO₄) and concentrated to give a brownfoam which was purified using a Biotage Isolera automated chromatographysystem under normal phase conditions (silica column, gradient of 0→100%ethyl acetate in dichloromethane and then 0→50% methanol in ethylacetate) with detection at 254 nm to give3′-O-pivaloyl-5′-O-(tert-butyldimethylsilyl)-7-deaza-7-iodo-2′-deoxyguanosine(0.63 g, 68%), as a brown foam.

R_(f) 0.41 (dichloromethane-ethyl acetate, 40:60, v/v).

¹H NMR (300 MHz, DMSO-d₆) δ 10.53 (s, 1H, NH), 7.09 (s, 1H, CH), 6.39(br s, 2H, NH₂), 6.26 (dd, J=9.0, 5.7 Hz, 1H, CHN), 5.14 (m, 1H, CH),3.95 (m, 1H, CH), 3.76 (m, 2H, CH₂), 2.5 (obscured m, 1H, 0.5×CH₂), 2.29(m, 1H, 0.5×CH₂), 1.17 (s, 9H, tert-butyl), 0.90 (s, 9H, TBDMStert-butyl), 0.10 (s, 3H, TBDMS CH₃), 0.08 (s, 3H, TBDMS CH₃).

3′-O-Pivaloyl-7-deaza-7-iodo-2′-deoxyguanosine

To an ice-cold solution of3′-O-pivaloyl-5′-O-(tert-butyldimethylsilyl)-7-deaza-7-iodo-2′-deoxyguanosine(100 mg, 0.17 mmol) in anhydrous THF (3 mL) was added 1 M TBAF in THF(0.25 mL, 0.25 mmol) dropwise and the mixture was stirred at roomtemperature for 1 h. The resulting solution was diluted with ethylacetate (10 mL) and the reaction mixture was quenched with water (10mL). The layers were separated and the aqueous layer was extracted withethyl acetate (2×10 mL) and chloroform (5 mL). The combined organic weredried (MgSO₄) and concentrated to give a yellow solid. This material waspurified using a Biotage Isolera automated chromatography system undernormal phase conditions (silica column, gradient of 0→80% methanol inethyl acetate) with detection at 254 nm to give3′-O-pivaloyl-7-deaza-7-iodo-2′-deoxyguanosine (77 mg, 95%), as a whitesolid.

R_(f) 0.60 (ethyl acetate-methanol, 9:1, v/v).

¹H NMR (300 MHz, DMSO-d₆) δ 10.51 (s, 1H, NH), 7.20 (s, 1H, CH), 6.36(br s, 2H, NH₂), 6.24 (m, 1H, CHN), 5.28 (m, 1H, CH) 5.11 (t, J=5.4 Hz,1H, OH) 3.95 (m, 1H, CH), 3.76 (m, 2H, CH₂), 2.60 (obscured m, 1H,0.5×CH₂), 2.23 (m, 1H, 0.5×CH₂), 1.18 (s, 9H, tert-butyl).

3′-O-Pivaloyl-7-deaza-7-(3-Boc-aminoprop-1-yn-1-yl)-2′-deoxyguanosine

A solution of 3′-O-pivaloyl-7-deaza-7-iodo-2′-deoxyguanosine (75 mg,0.16 mmol), tetrakis(triphenylphosphine)palladium (18 mg, 0.02 mmol) andcopper iodide (6 mg, 0.03 mmol) in anhydrous DMF (5 mL) was deoxygenatedfor 10 min. N-Boc-propargylamine (73 mg, 0.47 mmol) and Hunig's base (41mg, 55 μL, 0.31 mmol) were added, the solution was deoxygenated for afurther 10 min and the reaction mixture was stirred overnight in thedark at room temperature. The resulting mixture was diluted with ethylacetate (10 mL) and washed with 5% aqueous EDTA solution (10 mL). Thelayers were separated and the aqueous phase was extracted with ethylacetate (2×15 mL). The combined organic layers were washed with water(2×15 mL), saturated brine (20 mL), dried (MgSO₄) and concentrated togive a brown foam. This material was purified using a Biotage Isoleraautomated chromatography system under normal phase conditions (silicacolumn, gradient of 5→80% methanol in dichloromethane) with detection at254 nm to give3′-O-pivaloyl-7-deaza-7-(3-Boc-aminoprop-1-yn-1-yl)-2′-deoxyguanosine(57 mg, 72%), as an brown solid.

R_(f) 0.37 (dichloromethane-methanol, 93:7, v/v).

¹H NMR (300 MHz, DMSO-d₆) δ 10.56 (s, 1H, NH), 7.31 (m, 2H, CH+NH), 6.36(br s, 2H, NH₂), 6.25 (dd, J=9.5, 5.9 Hz, 1H, CHN), 5.25 (m, 1H, CH),5.13 (t, 1H, J=5.6 Hz, OH), 3.90 (m, 2H, CH₂), 3.55 (m, 2H, CH₂), 2.5(obscured m, 1H, 0.5×CH₂), 2.25 (m, 1H, 0.5×CH₂), 1.39 (s, 9H, Boctert-butyl), 1.17 (s, 9H, tert-butyl).

3′-O-Pivaloyl-7-deaza-7-(3-aminoprop-1-yn-1-yl)-2′-deoxyguanosinetrifluoroacetate

3′-O-Pivaloyl-7-deaza-7-(3-Boc-aminoprop-1-yn-1-yl)-2′-deoxyguanosine(85 mg, 0.17 mmol) was dissolved in anhydrous dichloromethane (1.25 mL)and trifluoroacetic acid (260 μL, 3.38 mmol) was added dropwise. Themixture was stirred for 2 h at room temperature and was concentratedunder reduced pressure. The residual trifluoroacetic acid wasazeotropically removed with chloroform (2×10 mL) and diethyl ether (2×10mL). The impure material was purified using a Biotage Isolera automatedchromatography system under reversed-phase conditions (C₁₈ column,gradient of 0→36% acetonitrile in 0.1% aqueous trifluoroacetic acid)with detection at 254 nm to afford, after freeze-drying,3′-O-pivaloyl-7-deaza-7-(3-aminoprop-1-yn-1-yl)-2′-deoxyguanosinetrifluoroacetate (Fragment 1) (60 mg, 69%), as an off-white solid.

¹H NMR (300 MHz, DMSO-d₆) δ 10.64 (s, 1H, NH), 8.25 (br s, 3H, NH₃ ⁺),7.39 (s, 1H, CH), 6.43 (br s, 2H, NH₂), 6.27 (dd, J=9.1, 5.8 Hz, 1H,CHN), 5.27 (m, 1H, CH), 3.96 (m, 3H, CH+CH₂), 3.58 (m, 2H, CH₂), 2.62(m, 1H, 0.5×CH₂), 2.25 (m, 1H, 0.5×CH₂), 1.18 (s, 9H, tert-butyl).

3-[(1,3)Dioxolan-2-ylmethoxy]benzoic acid ethyl ester

Ethyl 3-hydroxybenzoate (3.50 g, 21.1 mmol), bromoethyl-1,3-dioxolane(14.1 g, 8.72 mL, 84.3 mmol), potassium carbonate (5.83 g, 42.1 mmol)and sodium iodide (1.26 g, 8.43 mmol) were dissolved in anhydrous DMF(10 mL) and the reaction mixture was stirred at 120° C. for 20 h. Thesuspension was cooled to room temperature and quenched with water (30mL). The aqueous phase was extracted with ethyl acetate (3×50 mL). Theorganic layers were combined, washed with water (5×100 mL) and saturatedbrine (100 mL), before being dried (MgSO₄) and concentrated underreduced pressure to afford an orange oil. This residual material waspurified using a Biotage Isolera automated chromatography system undernormal phase conditions (silica column, gradient of 0→30% ethyl acetatein petrol) with detection at 254 nm to give3-[(1,3)dioxolan-2-ylmethoxy]benzoic acid ethyl ester (5.09 g, 96%), asa colorless oil.

R_(f) 0.40 (petrol-ethyl acetate, 75:25, v/v).

¹H NMR (400 MHz, CDCl₃): δ 7.66 (dt, J=7.5, 1.1 Hz, 1H, ArH), 7.59 (dd,J=2.6, 1.5 Hz, 1H, ArH), 7.34 (t, J=7.9 Hz, 1H, ArH), 7.14 (ddd, J=8.3,2.6, 1.0 Hz, 1H, ArH), 5.31 (t, J=4.1 Hz, 1H, CH), 4.36 (q, J=7.2 Hz,2H, OCH₂), 4.02 (m, 6H, OCH₂CH₂O+ArOCH₂), 1.39 (t, J=7.1 Hz, 3H, CH₃).

3-[2-Azido-2-(2-hydroxyethoxy)ethoxy]benzoic acid ethyl ester

To a mixture of 3-[(1,3)dioxolan-2-ylmethoxy]benzoic acid ethyl ester(5.08 g, 20.1 mmol) and azidotrimethylsilane (2.55 g, 2.94 mL, 22.2mmol) was added tin(IV) chloride (336 mg, 151 μL, 1.29 mmol). Thereaction mixture was stirred at room temperature for 2 h. The resultingmixture was diluted with 2% aqueous methanol (60 mL) and stirred for 30min before being concentrated under reduced pressure. The residue wasazeotropically dried with ethanol (2×30 mL) to afford a colorless oil.This oil was purified using a Biotage Isolera automated chromatographysystem under normal phase conditions (silica column, gradient of 0→70%ethyl acetate in petrol) with detection at 254 nm to give3-[2-azido-2-(2-hydroxyethoxy)ethoxy]benzoic acid ethyl ester (3.59 g,59%), as a colorless oil.

R_(f) 0.41 (petrol-ethyl acetate, 53:47, v/v).

¹H NMR (300 MHz, CDCl₃): δ 7.69 (dt, J=7.7, 1.2 Hz, 1H, ArH), 7.59 (dd,J=2.6, 1.5 Hz, 1H, ArH), 7.36 (t, J=8.0 Hz, 1H, ArH), 7.13 (ddd, J=8.3,2.7, 1.0 Hz, 1H, ArH), 4.89 (t, J=5.1 Hz, 1H, CH), 4.38 (q, J=7.1 Hz,2H, OCH₂), 4.19 (m, 2H, ArOCH₂), 4.00 (m, 1H, 0.5×OCH₂), 3.80 (m, 3H,0.5×OCH₂+OCH₂), 1.40 (t, J=7.1 Hz, 3H, CH₃).

3-[2-Azido-2-(2-hydroxyethoxy)ethoxy]benzoic acid

3-[2-Azido-2-(2-hydroxyethoxy)ethoxy]benzoic acid ethyl ester (3.51 g,11.9 mmol) was dissolved in ethanol (30 mL) and 4 M aqueous sodiumhydroxide (30 mL) was added. The mixture was stirred at room temperaturefor 3.5 h and the volume was then reduced by ¾ under vacuum. Theresulting mixture was diluted with water (50 mL) and acidified to pH 1-2with 2 M hydrochloric acid. This mixture was extracted withdichloromethane (3×100 mL). The combined organic phases were washed withsaturated brine (150 mL), dried (MgSO₄) and concentrated under reducedpressure to afford 3-[2-azido-2-(2-hydroxyethoxy)ethoxy]benzoic acid(3.39 g, quantitative), as a colorless oil which was used withoutfurther purification.

¹H NMR (300 MHz, CDCl₃): δ 7.76 (dt, J=7.7, 1.2 Hz, 1H, ArH), 7.65 (dd,J=2.4, 1.4 Hz, 1H, ArH), 7.41 (t, J=8.0 Hz, 1H, ArH), 7.19 (ddd, J=8.2,2.7, 1.0 Hz, 1H, ArH), 4.90 (t, J=5.1 Hz, 1H, CH), 4.21 (m, 2H, ArOCH₂),4.03 (m, 1H, 0.5×OCH₂), 3.81 (m, 3H, 0.5×OCH₂+OCH₂).

3-[2-Azido-2-(2-ethoxycarbonylmethoxyethoxy)ethoxy]benzoic acid

To an ice-cold solution of 3-[2-azido-2-(2-hydroxyethoxy)ethoxy]benzoicacid (1.42 g, 5.31 mmol) in anhydrous THF (18 mL) was added sodiumhydride (0.64 g, 15.9 mmol) and the mixture was stirred at 0° C. for 10min. Ethyl bromoacetate (1.95 g, 1.30 mL, 11.7 mmol) was added and thereaction mixture was allowed to warm up to room temperature over 5 h.The resulting mixture was quenched with ice-water (20 mL) and washedwith dichloromethane (2×70 mL). The aqueous layer was acidified to pH1-2 using 2 M hydrochloric acid and was extracted with dichloromethane(3×70 mL). The combined organic layers were dried (MgSO₄) andconcentrated under reduced pressure to afford a colorless oil. Thismaterial was purified using a Biotage Isolera automated chromatographysystem under normal phase conditions (silica column, gradient of 0→60%ethyl acetate in dichloromethane) with detection at 254 nm to give3-[2-azido-2-(2-ethoxycarbonylmethoxyethoxy)ethoxy]benzoic acid (0.62 g,33%), as a colorless oil.

R_(f) 0.45 (ethyl acetate-dichloromethane, 4:6 v/v).

¹H NMR (300 MHz, CDCl₃): δ 7.75 (dt, J=7.7, 1.2 Hz, 1H, ArH), 7.64 (dd,J=2.7, 1.5 Hz, 1H, ArH), 7.40 (t, J=8.0 Hz, 1H, ArH), 7.19 (ddd, J=8.3,2.7, 1.0 Hz, 1H, ArH), 4.95 (dd, J=5.5, 4.6 Hz, 1H, CH), 4.14 (m, 7H,ArOCH₂+0.5×OCH₂+OCH₂+OCH₂C(O)), 3.86 (m, 3H, 0.5×OCH₂+OCH₂), 1.28 (t,J=7.2 Hz, 3H, CH₃).

N-(2-Aminoethyl)-2,2,2-trifluoroacetamide Trifluoroacetate

To an ice-cold solution of N-Boc-ethylenediamine (2.08 g, 2.05 mL, 13.0mmol) in anhydrous THF (8 mL) was slowly added ethyl trifluoroacetate(1.85 g, 1.55 mL, 13.0 mmol) and the reaction mixture was stirred atroom temperature overnight. The resulting solution was concentratedunder reduced pressure to give tert-butyl[2-(2,2,2-trifluoroacetamido)ethyl]carbamate (3.30 g, 99%), as a whitesolid which was used without further purification.

tert-Butyl [2-(2,2,2-trifluoroacetamido)ethyl]carbamate (1.04 g, 4.06mmol) was dissolved in trifluoroacetic acid (5 mL) and stirred at roomtemperature for 30 min. The resulting mixture was concentrated underreduced pressure and the residual trifluoroacetic acid wasazeotropically removed with chloroform (3×10 mL). The material obtainedwas dried in vacuo at 50° C. for 2 h to giveN-(2-aminoethyl)-2,2,2-trifluoroacetamide trifluoroacetate (1.07 g,99%), as a yellow oil which was used without further purification.

¹H NMR (300 MHz, DMSO-d₆): δ 9.58 (br, 1H, NH), 7.99 (br, 3H, NH₃ ⁺),3.43 (m, 2H, CH₂N), 2.97 (m, 2H, CH₂N).

[2-(1-Azido-2-{3-[2-(2,2,2-trifluoroacetamido)ethylcarbamoyl]phenoxy}ethoxy)ethoxy]aceticacid ethyl ester

To a solution of N-(2-aminoethyl)-2,2,2-trifluoroacetamidetrifluoroacetate (1.31 g, 4.86 mmol) in anhydrous DMF (25 mL) was addedHunig's base (1.57 g, 2.12 mL, 12.1 mmol),3-[2-azido-2-(2-ethoxycarbonylmethoxyethoxy)ethoxy]benzoic acid (1.43 g,4.05 mmol) and PyBOP (2.32 g, 4.45 mmol). The reaction mixture wasstirred at room temperature for 20 h before being diluted with ethylacetate (20 mL) and quenched with 1 M hydrochloric acid (40 mL). Theaqueous phase was extracted with ethyl acetate (3×50 mL). The combinedorganic layers were washed with water (5×100 mL), saturated brine (100mL), dried (MgSO₄) and concentrated under reduced pressure to give ayellow oil. This oil was purified using a Biotage Isolera automatedchromatography system under normal phase conditions (silica column,gradient of 0→90% ethyl acetate in dichloromethane) with detection at254 nm to give[2-(1-azido-2-{3-[2-(2,2,2-trifluoroacetamido)ethylcarbamoyl]phenoxy}ethoxy)ethoxy]aceticacid ethyl ester (1.75 g, 88%), as a colorless oil.

R_(f) 0.48 (dichloromethane-ethyl acetate, 1:1, v/v).

¹H NMR (300 MHz, CDCl₃): δ 8.01 (br, 1H, NH), 7.39 (m, 3H, 3×ArH), 7.10(dt, J=5.8, 3.0 Hz, 1H, ArH), 6.97 (br, 1H, NH), 4.93 (t, J=5.1 Hz, 1H,CH), 4.18 (m, 6H, ArOCH₂+OCH₂+OCH₂C(O)), 4.04 (m, 1H, 0.5×OCH₂), 3.87(m, 1H, 0.5×OCH₂), 3.81 (m, 2H, OCH₂), 3.69 (m, 2H, CH₂N), 3.62 (m, 2H,CH₂N), 1.30 (t, J=7.1 Hz, 3H, CH₃).

Sodium(2-{2-[3-(2-aminoethylcarbamoyl)phenoxy]-1-azidoethoxy}ethoxy)acetate

[2-(1-Azido-2-{3-[2-(2,2,2-trifluoroacetamido)ethylcarbamoyl]phenoxy}ethoxy)ethoxy]aceticacid ethyl ester (0.90 g, 1.83 mmol) was dissolved in ethanol (7 mL) and4 M aqueous sodium hydroxide (7 mL) was added. The resulting mixture wasstirred at room temperature for 2.5 h before being concentrated underreduced pressure. The material obtained was dissolved in water (55 mL)and washed with dichloromethane (2×48 mL). The aqueous layer wasacidified to pH 2 using 1 M hydrochloric acid and washed withdichloromethane (2×50 mL). The organic layer was removed and discardedwhile the aqueous layer was neutralised to pH 8 using 1 M aqueous sodiumhydroxide and evaporated under reduced pressure to give a white solidwhich was entrained with dichloromethane and methanol (2×95 mL, 1:1,v/v). The solid obtained was removed by suction filtration. The combinedfiltrates were concentrated under reduced pressure to give a gum. Thisimpure gum was entrained with dichloromethane and methanol (10 mL, 9:1,v/v) and the insoluble white solid obtained was removed by suctionfiltration. The filtrate was concentrated under reduced pressure to givesodium(2-{2-[3-(2-aminoethylcarbamoyl)phenoxy]-1-azidoethoxy}ethoxy)acetate(0.76 g, quantitative), as a white foam.

¹H NMR (300 MHz, D₂O): δ 7.31 (m, 3H, 3×ArH), 7.11 (ddd, J=7.8, 2.6, 1.5Hz, 1H, ArH), 4.99 (t, J=4.5 Hz, 1H, CH), 4.15 (m, 2H, ArOCH₂), 3.94 (m,1H, 0.5×OCH₂), 3.79 (m, 3H, 0.5×OCH₂+OCH₂C(O)), 3.58 (m, 4H, OCH₂+CH₂N),3.10 (t, J=5.8 Hz, 2H, CH₂N).

2-(2-{1-Azido-2-[3-({2-[3,5-bis(methoxycarbonyl)benzamido]ethyl}carbamoyl)phenoxy]ethoxy}ethoxy)aceticacid (Fragment 2)

Trimethyl 1,3,5-benzenetricarboxylate (2.00 g, 7.93 mmol) was suspendedin methanol (180 mL) and 1 M aqueous NaOH solution (7.14 mL) was added.The resulting mixture was stirred at room temperature for 18 h. Thesolution was concentrated under reduced pressure to afford a white solidwhich was partitioned between dichloromethane (150 mL) and saturatedaqueous sodium bicarbonate solution (150 mL). The organic phase wasseparated and was extracted with saturated aqueous sodium bicarbonatesolution (150 mL) before being discarded. The combined aqueous layerswere acidified to pH 1-2 using concentrated hydrochloric acid and wereextracted with ethyl acetate (2×150 mL). The combined organic layerswere dried (MgSO₄) and concentrated to give3,5-bis(methoxycarbonyl)benzoic acid (1.66 g, 88%), as a white solid.

3,5-bis(Methoxycarbonyl)benzoic acid (0.80 g, 3.36 mmol) was dissolvedin ethyl acetate (20 mL) and N-hydroxysuccinimide (425 mg, 3.69 mmol)was added followed by N,N′-dicyclohexylcarbodiimide (0.76 g, 3.69 mmol).The resulting mixture was stirred at room temperature for 20 h. Thesuspension was filtered over Celite and the filtrate was washed withsaturated aqueous sodium bicarbonate solution (2×100 mL), water (100 mL)and saturated brine (100 mL). The organic layer was dried (MgSO₄) andconcentrated under reduced pressure to afford a white glassy solid. Thismaterial was purified using a Biotage Isolera automated chromatographysystem under normal phase conditions (silica column, gradient of 0→100%ethyl acetate in petrol) with detection at 254 nm to giveN-hydroxysuccinimido 3,5-bis(methoxycarbonyl)benzoate, (1.06 g, 95%) asa white solid.

R_(f) 0.44 (petrol-ethyl acetate, 4:6, v/v).

To a solution of N-hydroxysuccinimido 3,5-bis(methoxycarbonyl)benzoate(0.95 g, 2.83 mmol) and sodium(2-{2-[3-(2-aminoethylcarbamoyl)phenoxy]-1-azidoethoxy}ethoxy)acetate(0.92 g, 2.36 mmol) in anhydrous DMF (15 mL) was added Hunig's base(0.61 g, 0.83 mL, 4.72 mmol) and the reaction mixture was stirred atroom temperature overnight. The resulting mixture was diluted with ethylacetate (20 mL) and quenched with 1 M hydrochloric acid (20 mL). Theaqueous phase was separated and was extracted with ethyl acetate (3×30mL) and the combined organic phases were washed with water (5×50 mL) andsaturated brine (50 mL). The resulting solution was dried (MgSO₄) beforebeing concentrated under reduced pressure to give a white glassy solid.This material was purified using a Biotage Isolera automatedchromatography system under normal phase conditions (silica column,gradient of 0→50% methanol in dichloromethane) with detection at 254 nmto give2-(2-{1-azido-2-[3-({2-[3,5-bis(methoxycarbonyl)benzamido]ethyl}carbamoyl)phenoxy]ethoxy}ethoxy)aceticacid (Fragment 2) (0.92 g, 66%), as a white glassy solid.

R_(f) 0.37 (dichloromethane-methanol, 83:17, v/v).

¹H NMR (300 MHz, d₆-DMSO) δ 9.42 (br s, 1H, NH), 8.99 (br s, 1H, NH),8.66 (d, J=1.5 Hz, 2H, 2×ArH), 8.53 (t, J=1.5 Hz, 1H, ArH), 7.50 (m, 1H,ArH), 7.43 (d, J=7.7 Hz, 1H, ArH), 7.33 (t, J=7.9 Hz, 1H, ArH), 7.07(dd, J=8.0, 2.4 Hz, 1H, ArH), 5.21 (t, J=5.0 Hz, 1H, CH), 4.24 (dd,J=10.6, 4.4 Hz, 1H, 0.5×ArOCH₂), 4.07 (dd, J=10.6, 5.9 Hz, 1H,0.5×ArOCH₂), 3.88 (m, 8H, OCH₂+2×OCH₃), 3.84 (m, 1H, 0.5×OCH₂), 3.77 (m,1H, 0.5×OCH₂), 3.62 (m, 2H, OCH₂C(O)), 3.40 (m, 4H, 2×CH₂N).

Pre-Cleavable SCGTP

3′-O-Pivaloyl-7-deaza-7-(3-aminoprop-1-yn-1-yl)-2′-deoxyguanosinetrifluoroacetate (152 mg, 0.29 mmol) was dissolved in anhydrous DMF (4mL). Hunig's base (138 mg, 186 μL, 1.07 mmol) followed by2-(2-{1-azido-2-[3-({2-[3,5-bis(methoxycarbonyl)benzamido]ethyl}carbamoyl)phenoxy]ethoxy}ethoxy)aceticacid (157 mg, 0.27 mmol) and HBTU (107 mg, 0.28 mmol) were added. Thereaction mixture was stirred at room temperature overnight. Theresulting mixture was diluted with ethyl acetate (10 mL) and washed with1 M hydrochloric acid (10 mL). The aqueous phase was separated andextracted with ethyl acetate (2×10 mL). The combined organic phases werewashed with water (4×20 mL), saturated aqueous sodium bicarbonatesolution (20 mL), saturated brine (20 mL), dried (MgSO₄) andconcentrated under reduced pressure to give an orange solid. This crudesolid was purified using a Biotage Isolera automated chromatographysystem under normal phase conditions (silica column, gradient of 4→26%methanol in dichloromethane) with detection at 254 nm to givePre-Cleavable SCGTP (197 mg, 73%), as an orange solid.

R_(f) 0.46 (dichloromethane-methanol, 92:8, v/v).

¹H NMR (300 MHz, DMSO-d₆): δ 10.54 (br, 1H, NH), 9.08 (br, 1H, NH), 8.68(d, J=1.6 Hz, 2H, 2×ArH), 8.63 (br t, 1H, NH), 8.58 (t, J=1.6 Hz, 1H,ArH), 8.22 (t, J=5.7 Hz, 1H, NH), 7.46 (m, 2H, 2×ArH), 7.37 (t, J=7.8Hz, 1H, ArH), 7.30 (s, 1H, deoxyguanosine CH), 7.13 (m, 1H, ArH), 6.35(br s, 2H, deoxyguanosine NH₂), 6.24 (dd, J=9.2, 5.6 Hz, 1H, CHN), 5.25(m, 1H, OH), 5.14 (m, 2H, CHOPiv+CHN₃), 4.23 (dd, J=10.4, 4.5 Hz, 1H,0.5×ArOCH₂), 4.13 (m, 3H, 0.5×ArOCH₂+0.5×OCH₂+CH), 3.88 (m, 11H,CH₂N+1.5×OCH₂+2×OCH₃), 3.68 (m, 2H, deoxyguanosine CH₂), 3.55 (m, 2H,OCH₂C(O)), 3.46 (m, 4H, 2×CH₂N), 2.59 (m, 1H, deoxyguanosine 0.5×CH₂),2.25 (m, 1H, deoxyguanosine 0.5×CH₂), 1.17 (s, 9H, 3×CH₃).

Penta-Triethylammonium Cleavable SCGTP

Pre-Cleavable SCGTP (185 mg, 0.19 mmol) was dissolved in 1,4-dioxane(0.57 mL) and anhydrous pyridine (191 μL) and the flask was evacuatedand purged with a nitrogen atmosphere three times. 1.0 M Salicylchlorophosphite solution in 1,4-dioxane (210 μL, 0.21 mmol) was addedand the reaction mixture was stirred for 10 min. 0.5 M Tributylammoniumpyrophosphate solution in anhydrous DMF (0.57 ml, 0.29 mmol) andtributylamine (148 mg, 190 μL, 0.80 mmol) were simultaneously added andthe reaction mixture was stirred for 15 min. 1% Iodine solution inpyridine and water (3.8 mL, 92:8 v/v) was added and the solution wasstirred for 15 min before being quenched with 5% aqueous sodiumthiosulfate solution (100 μL) and the mixture was concentrated underreduced pressure. The residual material obtained was purified using aBiotage Isolera automated chromatography system under reversed-phaseconditions (C₁₈ column, gradient of 0→40% acetonitrile in 0.1 M TEAA atpH 8) with detection at 272 nm to afford, after freeze-drying, impureprotected Cleavable SCGTP (166 mg, 58%) which was used without furtherpurification.

To a solution of protected Cleavable SCGTP (166 mg) in water (1.20 mL)was added 1 M aqueous sodium hydroxide (1.18 mL, 1.18 mmol) and themixture was stirred at room temperature for 40 min. Further 1 M aqueoussodium hydroxide (0.60 mL, 0.60 mmol) was added and the mixture wasstirred at for 1 h 40 min. 1 M Aqueous sodium hydroxide (0.20 mL, 0.20mmol) was added, the mixture was stirred for 30 min and the solution wasdiluted with 1 M aqueous triethylammonium bicarbonate (TEAB) solution(pH 8, 5 mL). The resulting solution was lyophilized overnight (220 mg)and the material was dissolved in 1 M aqueous triethylammoniumbicarbonate (TEAB) (2.0 mL) to give a concentration of 110 mg/mL. Thissolution was purified by semi-preparative HPLC injecting 80 μL portionsand collecting the eluent containing the pure substance. The combinedfractions were reduced in volume by removing the acetonitrile and mostof the water and finally lyophilized to give penta-triethylammoniumCleavable SCGTP (97 mg, 58%), as a white solid.

HPLC Conditions:

Column: Phenomenex Luna C18(2), 15 mm×250 mm

Solvent Gradient: 90% 0.1 M aqueous TEAB (pH 7.0) to 85.6% 0.1 M aqueousTEAB (pH 7.0) over 22 min with the balance being acetonitrile.

Flow Rate: 7.8 mL/min

Temp: 30° C.

Detection: UV at 290 nm

Under these conditions the product had a retention time of ca. 18-19.5min.

Example 8 Synthesis of Photocleavable Linkers Synthesis of a3-(hydroxymethyl)naphthalen-2-ol derivative(3-(Allyloxy)naphthalen-2-yl)methyl (2,5-dioxopyrrolidin-1-yl)carbonate

To a stirred suspension of methyl 3-hydroxy-2-naphthoate (498 mg, 2.47mmol) and potassium carbonate (683 mg, 4.94 mmol) in acetone (8.2 mL) ina vial was added allyl bromide (419 mg, 321 μL, 3.71 mmol). The vial wassealed and heated at 80° C. for 3.5 h. After cooling to roomtemperature, allyl bromide (419 mg, 321 μL, 3.71 mmol) and potassiumiodide (616 mg, 3.71 mmol) were added. The vial was sealed and heated at80° C. for a further 72 h. After cooling to room temperature, thereaction mixture was concentrated under reduced pressure and the residuedissolved in ethyl acetate (10 mL), washed with water (2×10 mL), 3 Msodium hydroxide (10 mL), saturated brine (10 mL), dried (MgSO₄) andconcentrated to give methyl 3-(allyloxy)-2-naphthoate (615 mg, 98%), asa yellow oil that was used without further purification.

A stirred solution of methyl 3-(allyloxy)-2-naphthoate (300 mg, 1.24mmol) in anhydrous THF (6.2 mL) was cooled in an ice-bath under nitrogenand a solution of 1.0 M DIBAL in hexane (2.73 mL, 2.73 mmol) was addeddropwise. Stirring was continued overnight during which time thereaction mixture was allowed to warm to room temperature. The resultingmixture was cooled to 0° C., quenched with saturated aqueous sodiumbicarbonate solution (5 mL) and diluted with ethyl acetate (10 mL) andwater (20 mL). The layers were separated and the aqueous layer wasextracted with ethyl acetate (2×10 mL). The combined organic layers werewashed with water (2×30 mL), saturated brine (10 mL), dried (MgSO₄) andconcentrated to give a yellow oil. This crude material was purifiedusing a Biotage Isolera automated chromatography system under normalphase conditions (silica column, gradient of 5→40% petrol in ethylacetate) with detection at 254 nm to afford(3-(allyloxy)naphthalen-2-yl)methanol (220 mg, 83%), as a yellow oil.

R_(f)=0.20 (ethyl acetate-petrol, 1:4 v/v)

To a stirred solution of (3-(allyloxy)naphthalen-2-yl)methanol (215 mg,1.00 mmol) in anhydrous acetonitrile (5 mL) under nitrogen was addedN,N′-disuccinimidyl carbonate (335 mg, 1.31 mmol) and pyridine (103 mg,106 μL, 1.31 mmol) and stirring was continued at room temperatureovernight. The mixture was evaporated to dryness and the residuedissolved in dichloromethane (20 mL), and washed with saturated aqueoussodium bicarbonate solution (3×20 mL), water (20 mL), saturated brine(20 mL), dried (MgSO₄) and concentrated to give(3-(allyloxy)naphthalen-2-yl)methyl (2,5-dioxopyrrolidin-1-yl)carbonate(263 mg, 74%), as a white solid that was used without furtherpurification.

(3-Hydroxynaphthalen-2-yl)methyl (2-(benzyloxy)-1-methoxyethyl)carbamate

A solution of triethylamine (111 mg, 153 μL, 1.10 mmol) in dioxane (1mL) was added to a slurry of H-Ser(OBn)-OH (143 mg, 0.73 mmol) in water(2 mL). Once a clear solution was obtained, a solution of(3-(allyloxy)naphthalen-2-yl)methyl (2,5-dioxopyrrolidin-1-yl)carbonate(260 mg, 0.73 mmol) in anhydrous dioxane (2 mL) was added and stirringwas continued at room temperature overnight. The solution was pouredinto water (6 mL), acidified to ˜pH 3.0 with 2.0 M aqueous sodiumbisulfate and extracted with ethyl acetate (3×30 mL). The combinedorganic layers were washed with water (3×30 mL), saturated brine (30mL), dried (MgSO₄) and concentrated to giveN-(((3-(allyloxy)naphthalen-2-yl)methoxy)carbonyl)-O-benzylserine (290mg, 91%), as a colorless oil that was used without further purification.

A stirred solution of lead(IV) acetate (357 mg, 0.80 mmol) in anhydrousDMF (0.75 mL) was cooled in an ice-bath under nitrogen and a solution ofN-(((3-(allyloxy)naphthalen-2-yl)methoxy)carbonyl)-O-benzylserine (290mg, 0.67 mmol) in anhydrous DMF (0.75 mL) was added dropwise. Stirringwas continued for 30 min at this temperature and for a further 4 hduring which time the reaction mixture was allowed to warm to roomtemperature. Ethyl acetate (10 mL) was added and the mixture wasquenched with saturated sodium bicarbonate solution (10 mL). The layerswere separated and the aqueous layer was extracted with ethyl acetate(3×10 mL). The combined organic layers were washed with saturatedaqueous sodium bicarbonate solution (10 mL), saturated brine (10 mL),dried (MgSO₄) and concentrated to afford1-((((3-(allyloxy)naphthalen-2-yl)methoxy)carbonyl)amino)-2-(benzyloxy)ethylacetate (326 mg), as a yellow oil.

1-((((3-(Allyloxy)naphthalen-2-yl)methoxy)carbonyl)amino)-2-(benzyloxy)ethylacetate (324 mg) was dissolved in anhydrous methanol (3.5 mL). Thesolution was heated under microwave irradiation at 100° C. for 30 minbefore being concentrated under reduced pressure. Residual acetic acidwas removed azeotropically with methanol (3×3 mL) to afford(3-(allyloxy)naphthalen-2-yl)methyl(2-(benzyloxy)-1-methoxyethyl)carbamate (276 mg, 98% over 2 steps), as ayellow/orange oil that was used without further purification.

To a stirred solution of (3-(allyloxy)naphthalen-2-yl)methyl(2-(benzyloxy)-1-methoxyethyl)carbamate (127 mg, 0.30 mmol) and5,5-dimethyl-1,3-cyclohexanedione (127 mg, 0.90 mmol) in anhydrous DMF(5 mL) under nitrogen was added Pd(PPh₃)₄ (104 mg, 0.09 mmol) andstirring was continued at room temperature overnight. The mixture wasevaporated to dryness and the residue taken up in water (10 mL) andextracted with ethyl acetate (5×10 mL). The combined organic layers werewashed with water (4×10 mL), saturated brine (10 mL), dried (MgSO₄) andconcentrated to give a dark orange sticky solid. This crude material wasinitially purified using a Biotage Isolera automated chromatographysystem under normal phase conditions (silica column, gradient of 0.5→4%methanol in dichloromethane) with detection at 254 nm to afford impure(3-hydroxynaphthalen-2-yl)methyl(2-(benzyloxy)-1-methoxyethyl)carbamate. Further purification using aBiotage Isolera automated chromatography system under normal phaseconditions (silica column, gradient of 0→1% methanol in diethyl ether)with detection at 254 nm gave (3-hydroxynaphthalen-2-yl)methyl(2-(benzyloxy)-1-methoxyethyl)carbamate (105 mg, 92%) as a pale brownsemi-solid.

R_(f)=0.80 (methanol-diethyl ether, 0.5:95.5 v/v)

Synthesis of a 2-hydroxymethyl-p-hydroquinone derivative2,5-Dimethoxybenzyl (2,5-dioxopyrrolidin-1-yl)carbonate

To 2,5-dimethoxybenzyl alcohol (0.67 g, 3.98 mmol) in anhydrousacetonitrile (20 mL) was added N,N′-disuccinimidyl carbonate (1.33 g,5.18 mmol) followed by pyridine (409 mg, 419 μL, 5.18 mmol) and themixture was stirred overnight at room temperature. The mixture wasconcentrated, reconstituted with dichloromethane (60 mL) and was washedwith saturated aqueous sodium bicarbonate (3×60 mL), water (60 mL),saturated brine (60 mL), dried (MgSO₄) and concentrated. The materialwas dried azeotropically with acetonitrile (3×20 mL) and diethyl ether(20 mL) to give 2,5-dimethoxybenzyl (2,5-dioxopyrrolidin-1-yl)carbonate(1.15 g, 93%), as a white solid.

Towards (3,6-Dioxocyclohexa-1,4-dien-1-yl)methyl(2-(benzyloxy)-1-methoxyethyl)carbamate

To O-benzylserine (0.73 g, 3.22 mmol) in water (10 mL) was addedtriethylamine (0.56 g, 0.78 mL, 5.58 mmol) in dioxane (10 mL) followedby 2,5-dimethoxybenzyl (2,5-dioxopyrrolidin-1-yl)carbonate (1.15 g, 3.22mmol) and the mixture was stirred overnight at room temperature. Theresulting mixture was diluted with water (30 mL) and was extracted withethyl acetate (3×50 mL). The combined organic layers were washed withwater (3×50 mL), saturated brine (50 mL), dried (MgSO₄) and concentratedto give O-benzyl-N-(((2,5-dimethoxybenzyl)oxy)carbonyl)serine (1.37 g,95%), as a white solid.

To lead (IV) tetraacetate (1.84 g, 4.16 mmol) in anhydrous DMF (4 mL) at−10° C. was added dropwiseO-benzyl-N-(((2,5-dimethoxybenzyl)oxy)carbonyl)serine (1.35 g, 3.47mmol) in anhydrous DMF (4 mL). The mixture was stirred at thistemperature for 30 min followed by room temperature overnight. Theresulting mixture was diluted with ethyl acetate (60 mL) and washed withsaturated aqueous sodium bicarbonate (60 mL). The aqueous layer wasextracted with ethyl acetate (30 mL) and the combined organic extractswere washed with saturated aqueous sodium bicarbonate (2×30 mL),saturated brine (30 mL), dried (MgSO₄) and concentrated to afford2-(benzyloxy)-1-((((2,5-dimethoxybenzyl)oxy)carbonyl)amino)ethyl acetate(1.38 g, 99%), as a yellow oil.

2-(Benzyloxy)-1-((((2,5-dimethoxybenzyl)oxy)carbonyl)amino)ethyl acetate(0.95 g, 2.35 mmol) in methanol (10 mL) was heated in a microwavereactor at 100° C. for 30 min. The resulting solution was concentratedand dried azeotropically with methanol (3×10 mL) to give2,5-dimethoxybenzyl (2-(benzyloxy)-1-methoxyethyl)carbamate (0.84 g,95%), as an orange oil.

Synthesis of a 2-Hydroxymethylanthraquinone Carbonate Derivative(9,10-Dioxo-9,10-dihydroanthracen-2-yl)methyl(2,5-dioxopyrrolidin-1-yl)carbonate

To a slurry of sodium acetate (1.50 g, 18.3 mmol) in glacial acetic acid(10 mL) and acetic anhydride (5 mL) was added2-bromomethyl-9,10-anthraquinone (0.50 g, 1.66 mmol) and the reactionmixture was heated at reflux overnight under nitrogen. After cooling toroom temperature, the solvent was evaporated and the residue wasslurried in water (10 mL) and collected by suction filtration using aBuchner funnel. This material was washed with water (2×10 mL) and driedin vacuo at room temperature overnight to afford2-methylacetate-9,10-anthraquinone (464 mg, quantitative), as a paleyellow solid.

A solution of sodium hydroxide (82 mg, 2.05 mmol) in water (2.3 mL) wasadded to a suspension of 2-methylacetate-9,10-anthraquinone (459 mg,1.64 mmol) and the mixture was heated at reflux for 4 h. After coolingto room temperature, the reaction mixture was diluted with water (18 mL)and the resulting solid collected by suction filtration. This materialwas washed with water (2×6 mL) and dried in vacuo at 40° C. overnight toafford 2-hydroxymethyl-9,10-anthraquinone (361 mg, 92%), as a paleyellow solid.

To a stirred solution of 2-hydroxymethyl-9,10-anthraquinone (358 mg,1.50 mmol) in anhydrous acetonitrile (7.5 mL) under nitrogen was addedN,N′-disuccinimidyl carbonate (500 mg, 1.95 mmol) and pyridine (154 mg,158 μL, 1.95 mmol) and stirring was continued at room temperatureovernight. The resulting mixture was evaporated to dryness and theresidue dissolved in dichloromethane (30 mL). The solution was washedwith saturated aqueous sodium bicarbonate solution (30 mL), the layerswere then separated and the aqueous layer was extracted with chloroform(4×30 mL). The combined organics were washed with saturated aqueoussodium bicarbonate solution (3×30 mL), water (30 mL), saturated brine(30 mL), dried (MgSO₄) and concentrated to afford(9,10-dioxo-9,10-dihydroanthracen-2-yl)methyl(2,5-dioxopyrrolidin-1-yl)carbonate (263 mg), as a pale yellow solidthat was used without further purification.

Further material was obtained by diluting the aqueous layer with ethylacetate (50 mL) and then collecting the resulting solid by suctionfiltration. This was dried in vacuo at room temperature for 72 h toafford (9,10-dioxo-9,10-dihydroanthracen-2-yl)methyl(2,5-dioxopyrrolidin-1-yl)carbonate (321 mg), as a pale yellow solidthat was used without further purification.

(9,10-Dioxo-9,10-dihydroanthracen-2-yl)methyl(2-(benzyloxy)-1-methoxyethyl)carbamate

A solution of triethylamine (126 mg, 174 μL, 1.25 mmol) in dioxane (3.4mL) was added to a slurry of H-Ser(OBn)-OH (162 mg, 0.83 mmol) in water(2.25 mL). Once a clear solution was obtained,(9,10-dioxo-9,10-dihydroanthracen-2-yl)methyl(2,5-dioxopyrrolidin-1-yl)carbonate (315 mg, 0.83 mmol) was added andstirring was continued at room temperature overnight. The solution waspoured into water (6 mL), acidified to ˜pH 3.0 with 2.0 M aqueous sodiumbisulfate and extracted with ethyl acetate (3×30 mL). The combinedorganic layers were washed with water (3×30 mL), saturated brine (30mL), dried (MgSO₄) and concentrated to affordO-benzyl-N-(((9,10-dioxo-9,10-dihydroanthracen-2-yl)methoxy)carbonyl)serine(236 mg, 62%), as a pale yellow solid.

A stirred solution of lead(IV) acetate (266 mg, 0.60 mmol) in anhydrousDMF (0.55 mL) was cooled in an ice-bath under nitrogen and a solution ofO-benzyl-N-(((9,10-dioxo-9,10-dihydroanthracen-2-yl)methoxy)carbonyl)serine(230 mg, 0.50 mmol) in anhydrous DMF (0.55 mL) was added dropwise.Stirring was continued for 30 min at this temperature followed by afurther 3 h during which time the reaction mixture was allowed to warmto room temperature. Ethyl acetate (10 mL) was added and the mixture wasquenched with saturated aqueous sodium bicarbonate solution (10 mL). Thelayers were separated and the aqueous layer was extracted with ethylacetate (3×10 mL). The combined organic layers were washed withsaturated aqueous sodium bicarbonate solution (10 mL), saturated brine(10 mL), dried (MgSO₄) and concentrated to give2-(benzyloxy)-1-((((9,10-dioxo-9,10-dihydroanthracen-2-yl)methoxy)carbonyl)amino)ethylacetate (270 mg), as a yellow oil.

2-(Benzyloxy)-1-((((9,10-dioxo-9,10-dihydroanthracen-2-yl)methoxy)carbonyl)amino)ethylacetate (270 mg) was dissolved in anhydrous methanol (2 mL) andanhydrous 1,2-dimethoxyethane (1.2 mL). The solution was heated undermicrowave irradiation at 100° C. for 30 min before being concentratedunder reduced pressure. Residual acetic acid was removed azeotropicallywith methanol (3×3 mL) to afford(9,10-dioxo-9,10-dihydroanthracen-2-yl)methyl(2-(benzyloxy)-1-methoxyethyl)carbamate (201 mg, 90% over 2 steps), as apale yellow solid.

Synthesis of a 2-Hydroxy-Substituted Linker Derivative1-((4-(tert-Butyl)phenoxy)carbonyl)-3-methyl-1H-imidazol-3-iumtetrafluoroboroate

To 4-tert-butylphenol (500 mg, 3.33 mmol) in anhydrous dichloromethane(10 mL) was added carbonyl diimidazole (567 mg, 3.50 mmol) and themixture was stirred overnight at room temperature. The impure materialwas dry-loaded onto silica and was purified using a Biotage Isoleraautomated chromatography system under normal phase conditions (silicacolumn, gradient of 7→77% ethyl acetate in petrol) with detection at 254nm to give 4-(tert-butyl)phenyl 1H-imidazole-1-carboxylate (461 mg,57%), as a white solid.

R_(f)=0.45 (ethyl acetate-petrol, 3:7 v/v)

4-(tert-Butyl)phenyl 1H-imidazole-1-carboxylate (50 mg, 0.21 mmol) andtrimethyloxonium tetrafluoroboroate (31 mg, 0.21 mmol) were stirred inanhydrous dichloromethane (1 mL) overnight at room temperature. Themixture was concentrated to give1-((4-(tert-butyl)phenoxy)carbonyl)-3-methyl-1H-imidazol-3-iumtetrafluoroboroate (55 mg), which was used without purification.

5-(2-(((4-(tert-Butyl)phenoxy)carbonyl)oxy)acetyl)-2-hydroxybenzoic acid

Benzyl bromide (2.49 g, 1.65 mL, 6.94 mmol) was added dropwise to asolution of 5-acetyl-2-hydroxybenzoic acid (1.00 g, 5.56 mmol) andpotassium carbonate (1.98 g, 6.94 g) in anhydrous DMF (15 mL) and themixture was stirred for 16 h at 120° C. The mixture was allowed to coolto room temperature and was diluted with water (15 mL) and extractedwith ethyl acetate (4×15 mL). The combined organic layers were washedwith water (5×15 mL), saturated brine (15 mL), dried (MgSO₄) andconcentrated. The impure residue was purified using a Biotage Isoleraautomated chromatography system under normal phase conditions (silicacolumn, gradient of 5→100% ethyl acetate in petrol) with detection at254 nm to give benzyl 5-acetyl-2-(benzyloxy)benzoate (1.86 g, 93%), asan off-white solid.

R_(f)=0.45 (ethyl acetate-petrol, 1:4 v/v)

Phenyltrimethylammonium tribromide (PTT) (1.56 g, 4.16 mmol) was addedportionwise to benzyl 5-acetyl-2-(benzyloxy)benzoate (1.50 g, 4.16 mmol)in anhydrous THF (15 mL) and the mixture was stirred for 20 min.Ice-cold water (90 mL) was added and crystallisation was induced bystoring at 4° C. The crystals were collected by suction filtration usinga Buchner funnel and were washed with diethyl ether (20 mL) to givebenzyl 2-(benzyloxy)-5-(2-bromoacetyl)benzoate (1.51 g, 83%), as anoff-white crystalline solid after drying in vacuo for 1 h at 40° C.

To a suspension of benzyl 2-(benzyloxy)-5-(2-bromoacetyl)benzoate (1.51g, 3.44 mmol) in ethanol (4 mL) was added sodium acetate (310 mg, 3.78mmol) in water (2 mL), and acetic acid (200 μL) and the mixture wasstirred for 3 h at 80° C. The resulting mixture was allowed to cool toroom temperature and crystallisation was induced by storing at 4° C. Thecrystals were collected by suction filtration using a Buchner funnel andwere washed sequentially with ice-cold water (10 mL) and ice-colddiethyl ether (10 mL) to afford5-(2-acetoxyacetyl)-2-(benzyloxy)benzoate (707 mg, 49%), as a whitecrystalline solid after drying in vacuo for 1 h at 40° C.

To benzyl 5-(2-acetoxyacetyl)-2-(benzyloxy)benzoate (300 mg, 0.72 mmol)in methanol (3 mL) was added 2 M hydrochloric acid (1.8 mL) and thesolution was stirred at 70° C. for 1 h. The resulting mixture wasallowed to cool to room temperature and was diluted with water (20 mL)and extracted into ethyl acetate (20 mL). The organic layer was dried(MgSO₄) and concentrated. The impure material was purified using aBiotage Isolera automated chromatography system under normal phaseconditions (silica column, gradient of 12→85% ethyl acetate in petrol)with detection at 254 nm to give benzyl2-(benzyloxy)-5-(2-hydroxyacetyl)benzoate (225 mg, 83%), as a whitesolid.

R_(f)=0.42 (ethyl acetate-petrol, 1:1 v/v)

To benzyl 2-(benzyloxy)-5-(2-hydroxyacetyl)benzoate (80 mg, 0.21 mmol)in anhydrous THF (2 mL) was added1-((4-(tert-butyl)phenoxy)carbonyl)-3-methyl-1H-imidazol-3-iumtetrafluoroboroate (55 mg, 0.21 mmol) and the mixture was stirred atroom temperature overnight. TLC showed that the reaction was incompleteand the mixture was heated at reflux for 5 h. The impure material wasdry-loaded onto silica and was purified using a Biotage Isoleraautomated chromatography system under normal phase conditions (silicacolumn, gradient of 10→100% ethyl acetate in petrol) with detection at254 nm to give benzyl2-(benzyloxy)-5-(2-(((4-(tert-butyl)phenoxy)carbonyl)oxy)acetyl)benzoate(88 mg, 80%), as a colorless oil.

R_(f)=0.45 (ethyl acetate-petrol, 1:1 v/v)

10% Palladium on carbon (8.5 mg) was cautiously wetted with ethylacetate (2 mL), under nitrogen, and benzyl2-(benzyloxy)-5-(2-(((4-(tert-butyl)phenoxy)carbonyl)oxy)acetyl)benzoate(85 mg, 0.16 mmol) was added. An atmosphere of hydrogen was introducedvia a balloon and the mixture was stirred for 2 h at room temperature.The catalyst was removed by filtration of the suspension through a thinlayer of Celite and the filtrate was concentrated to give5-(2-(((4-(tert-butyl)phenoxy)carbonyl)oxy)acetyl)-2-hydroxybenzoic acid(60 mg, 100%), as a white solid.

Example 9 Synthesis of Chemically Cleavable Linkers Methyl2-(azidomethyl)benzoate

To methyl 2-(bromomethyl)benzoate (250 mg, 1.09 mmol) in anhydrous DMF(4 mL) was added sodium azide (565 mg, 8.70 mmol) and the solution wasstirred at room temperature overnight. The resulting solution wasdiluted with water (40 mL) and extracted into ethyl acetate (3×50 mL).The combined organic layers were washed with water (5×40 mL), saturatedbrine (40 mL), dried (MgSO₄) and concentrated to give methyl2-(azidomethyl)benzoate (203 mg, 97%), as a pale yellow solid.

2-(Azidomethyl)benzoic acid

To methyl 2-(azidomethyl)benzoate (200 mg, 1.04 mmol) in THF (1 mL) wasadded 3 M sodium hydroxide (1.5 mL) and the solution was stirred at roomtemperature overnight. The resulting solution was diluted with water (10mL) and washed with diethyl ether (3×10 mL). The aqueous layer wasadjusted to pH 2 with 2 M hydrochloric acid and subsequently extractedwith ethyl acetate (3×20 mL). The combined organic layers were washedwith saturated brine (10 mL), dried (MgSO₄) and concentrated to give2-(azidomethyl)benzoic acid (174 mg, 95%), as a white solid.

2-(Azidomethyl)benzoyl chloride

To 2-(azidomethyl)benzoic acid (170 mg, 0.96 mmol) and anhydrous DMF(3.5 mg, 4 μL, 0.05 mmol) in anhydrous THF (3 mL) was added thionylchloride (342 mg, 210 μL, 2.88 mmol) and the mixture was stirred at 60°C. for 2 h. The reaction mixture was allowed to cool to room temperatureand was concentrated to give 2-(azidomethyl)benzoyl chloride (181 mg,quantitative), as a yellow solid that was used immediately withoutpurification.

4-Nitrophenyl 2-(azidomethyl)benzoate

To 2-(azidomethyl)benzoyl chloride (90 mg, 0.48 mmol) and DMAP (117 mg,0.96 mmol) in anhydrous dichloromethane (1 mL) was added dropwise4-nitrophenol (74 mg, 0.53 mmol) in anhydrous dichloromethane (1 mL) andthe reaction mixture was stirred at room temperature overnight. Theresulting impure material was dry-loaded onto silica and was purifiedusing a Biotage Isolera automated chromatography system under normalphase conditions (silica column, gradient of 12→100% ethyl acetate inpetrol) with detection at 254 nm to give 4-nitrophenyl2-(azidomethyl)benzoate (89 mg, 55%), as a white solid.

R_(f)=0.54 (ethyl acetate-petrol, 1:1 v/v)

7-Hydroxy-4-methylcoumarinyl 2-(azidomethyl)benzoate

To 2-(azidomethyl)benzoyl chloride (90 mg, 0.48 mmol) and DMAP (117 mg,0.96 mmol) in anhydrous dichloromethane (1 mL) was added dropwise4-methylumbelliferone (93 mg, 0.53 mmol) in anhydrous dichloromethane (1mL) and the reaction mixture was stirred at room temperature overnight.The resulting impure material was dry-loaded onto silica and waspurified using a Biotage Isolera automated chromatography system undernormal phase conditions (silica column, gradient of 12→75% ethyl acetatein petrol) with detection at 254 nm to give 7-hydroxy-4-methylcoumarinyl2-(azidomethyl)benzoate (80 mg, 56%), as a white solid.

R_(f)=0.57 (ethyl acetate-petrol, 1:1 v/v)

What is claimed is:
 1. A reporter composition, comprising: a nucleotideor its derivative; a high charge mass moiety comprising an aromatic oraliphatic skeleton, comprising one or more charged groups selected fromthe group consisting of a tertiary amino group, a carboxyl group, ahydroxyl group, a phosphate group, a phenolic hydroxy group, anyderivatives thereof, and any combinations thereof, wherein the one ormore charged groups comprise a charge mass that is sufficient togenerate a detectable change in a property of a sensitive detectionnanostructure or nano-/micro-sensor operably coupled to the reportercomposition; and a linker molecule attached to the nucleotide or itsderivative and the high charge mass moiety, wherein the linker moleculecomprises a linear or branched chain comprising one or more selectedfrom the group consisting of an alkyl group, an oxy alkyl group, analcohol group, a carboxyl group, an amine group, an amide group, anaromatic group, and a naphthalene group, any derivatives thereof, andany combinations thereof.
 2. The reporter composition of claim 1,wherein the nucleotide is selected from the group consisting of adeoxyribonucleotide, a ribonucleotide, a peptide nucleotide, amorpholino, a locked nucleotide, a glycol nucleotide, a threosenucleotide, and a synthetic nucleotide, and any isoforms thereof.
 3. Thereporter composition of claim 1, wherein the high charge mass moietycomprises one or more selected from the group consisting of thefollowing compounds 1-5, any derivatives thereof, and any combinationsthereof:


4. The reporter composition of claim 1, wherein the linker molecule isphotocleavable or chemically cleavable.
 5. The reporter composition ofclaim 4, wherein the chemically cleavable linker molecule comprises oneor more selected from the group consisting of: 4-Nitrophenyl2-(azidomethyl)benzoate, 7-Hydroxy-4-methylcoumarinyl2-(azidomethyl)benzoate, any derivatives thereof, and any combinationsthereof.
 6. The reporter composition of claim 1, wherein thephotocleavable cleavable linker molecule comprises one or more selectedfrom the group consisting of the following compounds 6-10, anyderivatives thereof, and any combinations thereof:


7. The reporter composition of claim 1, wherein a number of an alkylgroup, an oxy alkyl group, an alcohol group, a carboxyl group, an aminegroup, am amide group, an aromatic group, and a naphthalene group, anyderivatives thereof, and any combinations thereof in the linear orbranched chain is 1 to
 1000. 8. The reporter composition of claim 1,wherein the linker molecule or the high charge mass moiety is configurednot to affect nucleotide polymerization by a polymerase.
 9. The reportercomposition of claim 1, wherein the high charge mass moiety isconfigured to extend out from a nucleotide polymerase complex.
 10. Thereporter composition of claim 1, wherein the linker molecule or the highcharge mass moiety is configured to protrude out from a nascent chain soas to reach-down toward the sensitive detection nanostructure or sensor.11. The reporter composition of claim 1, wherein a net charge mass ofthe high charge mass moiety is positive or negative, or is variabledepending on pH.
 12. The reporter composition of claim 1, wherein a netcharge mass of the high charge mass moiety is positive in turn in anacidic pH.
 13. The reporter composition of claim 1, wherein a net chargemass of the high charge mass moiety is negative in turn in an alkalinepH.
 14. The reporter composition of claim 1, wherein the linker moleculeand/or the high charge mass moiety is configured to be removable. 15.The reporter composition of claim 1, wherein the reporter compositioncomprises one or more selected from the group consisting of thefollowing compounds, any derivatives thereof, and any combinationsthereof:


16. A kit for determining a nucleotide sequence, comprising the reportercomposition of claim
 1. 17. A method of synthesizing the reportercomposition of claim 1, comprising: generating a first covalent linkagebetween the nucleotide and a first functional group of the linker,wherein a phosphate group, a sugar or a base of the nucleotide is linkedto the first functional group of the linker; and generating a secondcovalent linkage between a second functional group of the linker and anyfunctional group present in the high charge mass moiety.
 18. The methodof claim 17, wherein the nucleotide comprises a triphosphate group,selected from the group consisting of adenosine triphosphate (ATP),guanosine triphosphate (GTP), cytidine triphosphate (CTP), thymidinetriphosphate (TTP), and uridine triphosphate (UTP).
 19. The method ofclaim 17, wherein the phosphate group of the nucleotide comprisesthiophosphate or phosphoramidate.